Stable forms of alpha-synuclein oligomers

Abstract

There is provided a mutant form of alpha-synuclein. For example the mutation may be selected from a) the substitution or insertion of a proline residue in an amino acid position that corresponds to residues 26-37 of SEQ ID NO 1, b) the substitution or insertion of a proline residue in an amino acid in an amino acid position that corresponds to residues 54-65 of SEQ ID NO 1, or c) the substitution or insertion of a first cysteine residues in a first amino acid position that corresponds to positions 35 to 43 in SEQ ID NO 1 and the substitution or insertion of a second cysteine residue in a second amino acid position that corresponds to positions 48 to 56 in SEQ ID NO 1, where the first and second cysteine residues are at the same distance from a position corresponding to the bond between a position corresponding to residues 45 and 46 in SEQ ID NO 1, or where distances from each of the cysteines to a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1 differs with at most two residues.

Description

[0001] Stable forms of alpha-synuclein oligomers

[0002] Field of the invention

[0003] This invention relates to mutant forms of the protein alpha-synuclein. The mutant protein forms oligomers which can be used for screening for binding agents such as drug candidates for the treatment of Parkinson's disease.

[0004] Background

[0005] Parkinson's disease is a neurological disease characterized by the loss of dopaminergic neurons, which are responsible for motor control, cognitive function, etc.. Treatment of Parkinson's disease include administration of levodopa in order to lessens symptoms. However, there is a great need for improved treatment for Parkinson's.

[0006] The aggregation of alpha-synuclein is a central event in Parkinson's disease and other synucleinopathies (Spillantini, M.G., Goedert, M., 2000. Ann. N. Y Acad. Sci. 920, 16-27. https: / / doi.Org / 10.llll / j.1749-6632.2000.tb06900.x). The 140 amino acid protein is highly abundant in the presynapse (Wilhelm, B.G., et al., 2014. Science 344, 1023-1028. https: / / doi.org / 10.1126 / science.1252884) where it is thought to be involved in the regulation of synaptic vesicle trafficking and neurotransmitter release. Alpha-synuclein is intrinsically disordered in its cytosolic form whereas it adopts alpha-helical conformations upon binding to phospholipid membranes.

[0007] Under pathological conditions misfolded species of the protein accumulate. Somewhat simplified, the alpha-synuclein protein is present in three forms; monomers, various types of oligomers and fibrils. Despite extensive studies on the alpha-synuclein aggregation cascade from monomers converting to soluble oligomers and finally insoluble fibrils, the exact structure of the initial alpha-synuclein misfolding and early oligomers remains not completely understood yet due to their transient nature. Different studies have suggested the presence of a beta-hairpin formation in the region of residues 38-53 of the protein.

[0008] Alpha-synuclein oligomers convert into fibrils during which the oligomers need to undergo a conformational change (beta-hairpin opens up) to adopt the fibril cross-beta structure (Cremades, N., et al., 2012. Cell 149, 1048-1059. https: / / doi.Org / 10.1016 / j.cell.2012.03.037; Zhou, L., Kurouski, D., 2020. Anal. Chem. 92, 6806-6810. https: / / doi.org / 10.1021 / acs.analchem.0c00593).

[0009] Alpha-synuclein oligomers, in particular small alpha-synuclein oligomers, are considered to be the most toxic species of alpha-synuclein aggregates and are considered more toxic than large oligomers and fibrils (Cascella, R., et al., 2021. Nat Commun 12, 1814. https: / / doi.org / 10.1038 / s41467-021-21937-3; Emin, D., et al., 2022. Nat Commun 13, 5512. https: / / doi.org / 10.1038 / s41467-022-33252-6; Winner, B., et al., 2011. PNAS 108, 4194-4199. https: / / doi.org / 10.1073 / pnas.1100976108).

[0010] Hence, alpha-synuclein oligomers is a suitable target for an antibody-based drug for the treatment of Parkinsons.

[0011] However, alpha-synuclein oligomers are difficult to study because of their transient nature, quickly aggregating into fibrils (Chen, S.W., et al., 2015. Proc Natl Acad Sci U S A 112, E1994-2003. https: / / doi.org / 10.1073 / pnas.1421204112) or disintegrating into monomers (Mysling, S., et al., 2013. Biochemistry 52, 9097-9103. https: / / doi.org / 10.1021 / bi4009193). The transient nature of these early oligomers makes antibody development against a specific epitope on those oligomers nearly impossible.

[0012] There have therefore been different attempts at stabilizing alpha-synuclein oligomers. Previous attempts include the use of high protein concentration (Chen, S.W., et al., 2015. Proc Natl Acad Sci U S A 112, E1994-2003. https: / / doi.org / 10.1073 / pnas.1421204112; Froula, J.M., et al., 2019. JBC 294, 10392-10406. https : / / doi . org / 10 . 1074 / jbc . RAI 19 . 007743; Paslawski, W., et al., 2016. Methods Mol. Biol. Springer New York, New York, NY, pp. 133-150. https: / / doi.org / 10.1007 / 978-l-4939-2978-8_9), dopamine stabilization (Galkin, M., et al., 2023. ACS Chem. Neurosci. 14, 2027-2034. https: / / doi.org / 10.1021 / acschemneuro.2c00815; Planchard, M.S. et al., 2014. Protein Sci 23, 1369-1379. https: / / doi.org / 10.1002 / pro.2521), and crosslinking (Almandoz-Gil, L., et al., 2017. Free Radic Biol Med 110, 421-431. https: / / doi.org / 10.1016 / j.freeradbio- med.2017.07.004; Andersen, C., et al., 2021. Biochemistry 60, 3644-3658. https: / / doi.org / 10.1021 / acs.biochem.lc00478).

[0013] The previous methods for stabilizing alpha-synuclein oligomers have various disadvantages, including but not limited to: low yield, enrichment of undesired forms and incorrect (non-pathological or non-native) structure.

[0014] This has hampered the development of new drug candidates, including antibodies pecifi- cally targeting the pathological alpha-synuclein oligomers, for the treatment of Parkinson's and other disease related to alpha-synuclein.

[0015] Hence there is a need for an alpha-synuclein protein that provides high yield of therapeutically relevant structures and which can be used for the screening of binding agents.

[0016] This invention solves these and other problems.

[0017] Summary of the invention

[0018] The inventors have surprisingly found the introduction of mutations, in particular the introduction or substitution with proline or cysteine residues, results in the increased formation of oligomeric form of alpha-synuclein.

[0019] The invention provides alpha-synuclein mutants which show increased oligomer formation but which are resistant against fibrillation. The mutant protein according to the invention retain most of its wild-type like properties as detected by size exclusion chromatography (indicating same hydrodynamic radius on monomeric proteins), circular dichroism (indicating random coil structure in monomeric proteins) and mass photometry (indicating molecule mass distribution in oligomer samples

[0020] In a first aspect of the invention there is provided a mutant alpha-synuclein protein which has enhanced ability to form oligomers in vitro.

[0021] In various embodiments, the mutation is the substitution or insertion of a at least one pro- line or at least one cysteine residue. In various embodiments, the amino acid sequence of mutant alpha-synuclein has at least 80 % identity to SEQ ID NO 1, or at least 80% identity to a truncated version of SEQ ID NO 1 corresponding to at least residues 26 to 65 of SEQ ID NO 1.

[0022] In various embodiments, the molarity of an oligomer selected from a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, 9-mer, 10-mer, 20-mer, 30-mer or 50- mer of the alpha-synuclein protein is enriched in vitro with at least 50%, more preferably at least 75%, 100%, 200%, or 300 % compared to the wild-type protein as defined by SEQ ID NO 1. The molarity of the oligomer may for example be determined by size exclusion chromatography.

[0023] In various embodiments, the mutation is a mutation selected from a) the substitution or insertion of a proline residue in an amino acid position that corresponds to residues 26-37 of SEQ ID NO 1, b) the substitution or insertion of a proline residue in an amino acid position that corresponds to residues 54-65 of SEQ ID NO 1, or c) the substitution or insertion of a first cysteine residue in a first amino acid position that corresponds to positions 35 to 43 in SEQ ID NO 1 and the substitution or insertion of a second cysteine residue in a second amino acid position that corresponds to positions 48 to 56 in SEQ ID NO 1, where the first and second cysteine residues are at the same distance from a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1, or where the distances from each of the cysteines to a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1 differs with at most two residues.

[0024] In particular the mutations comprise alternatives a) and b) a) and b) and c) a) and c) or b) and c) in the paragraph above.

[0025] In particular the mutations may be

[0026] G36P and A56P,

[0027] G36P, V37C, T54C and A56P,

[0028] K34P, E35C, A56C and E57P, or

[0029] G36P, G41C, H50C and A56P. where the amino acid position corresponds to the amino acid positions in SEQ ID NO 1.

[0030] In various embodiments, the protein comprises or consists of one of SEQ ID NO 2 to SEQ ID NO 5.

[0031] In a second aspect of the invention there is provided a method for determining binding of a molecule to a pathogenic form of synuclein comprising contacting the molecule with a synuclein protein according to the first aspect of the invention and determining binding. The molecule may for example be an antibody or an antibody fragment.

[0032] In various embodiments, the method comprises contacting the molecule with a mutant synuclein protein in vitro and determining binding of the molecule to the synuclein protein. In various embodiments, synuclein protein is in oligomer form. In a third aspect of the invention there is provided a polynucleotide that codes for the protein of the first aspect of the invention.

[0033] In a fourth aspect of the invention there is provided a mutant alpha-synuclein protein where the amino acid sequence of the mutant alpha-synuclein has at least 80 % identity to SEQ ID NO 1, or at least 80% identity to a truncated version of SEQ ID NO 1 corresponding to at least residues 38 to 53, more preferably 26 to 65 of SEQ ID NO 1, where the mutation is one of a) the substitution or insertion of a proline residue in an amino acid position that corresponds to residues 26-37 of SEQ ID NO 1, b) the substitution or insertion of a proline residue in an amino acid in an amino acid position that corresponds to residues 54-65 of SEQ ID NO 1, or c) the substitution or insertion of a first cysteine residues in a first amino acid position that corresponds to positions 35 to 43 in SEQ ID NO 1 and the substitution or insertion of a second cysteine residue in a second amino acid position that corresponds to positions 48 to 56 in SEQ ID NO 1, where the first and second cysteine residues are at the same distance from a position corresponding to the bond between a position corresponding to residues 45 and 46 in SEQ ID NO 1, or where the distances from each of the cysteines to a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1 differs with at most two residues.

[0034] In various embodiments, the mutations comprise a) and b) or a) and b) and c) or a) and c) or b) and c). In various embodiments, the molarity of an oligomer selected from a dimer, tri- mer, tetramer, pentamer, hexamer, heptamer, octamer, 10-mer, 20-mer, 30-mer or 50-mer of the alpha-synuclein protein is enriched in vitro with at least 50% compared to the wild-type protein as defined by SEQ ID NO 1.

[0035] In various embodiments the molarity of the oligomer is determined by size exclusion chromatography.

[0036] In various embodiments fibril formation of the mutant protein is inhibited with at least 50 % compared to the wild type protein as defined by SEQ ID NO 1.

[0037] In various embodiments the mutation is at least

[0038] G36P and A56P,

[0039] G36P, V37C, T54C and A56P,

[0040] K34P, E35C, A56C and E57P, or

[0041] G36P, G41C, H50C and A56P, where the amino acid position corresponds to the amino acid positions in SEQ ID NO 1.

[0042] In various embodiments mutant synuclein protein comprises one of SEQ ID NO 2 to SEQ ID NO 5.

[0043] In various embodiments, the synuclein protein is n oligomer form.

[0044] Figure 1. Amino acid sequence alignment of wt alpha-synuclein and alpha-synuclein mutants in residues 34-57.

[0045] Figure 2. Seeded ThT assay with wt alpha-synuclein or alpha-synuclein mutants to monitor fibrillation, (a) wt alpha-synuclein, (b) P253, (c) P254, (d) P255, (e) P256. alpha-synuclein fibrillation monitored by ThT fluorescence (ex450 / em480) every 10 min. Wt alpha-synuclein or alpha-synuclein mutants at 40 pM were incubated with 0,1% PFF seeds in PBS at 37 °C with 5 min orbital shaking before each measurement. Note semi-logarithmic scale. An average of three replicates is shown.

[0046] Figure 3. Mass distribution analysis of alpha-synuclein soluble species after seeded aggregation. Supernatant fraction of (a) wt alpha-synuclein, (b) P253, (c) P254, (d) P255, (e) P256 after incubation under aggregation conditions like in Fig. 2 were analyzed by mass photometry.

[0047] Figure 4. Secondary structure analysis by circular dichroism (CD), (a) 0,2 mg / ml monomeric alpha-synuclein at pH 7,4. (b) Total sample after aggregation under conditions as in Fig. 2, diluted to 0,2 mg / ml. (c) Soluble species in supernatants of (b) after aggregation.

[0048] Figure 5. Co-incubation ThT assay with wt alpha-synuclein or alpha-synuclein mutants to monitor fibrillation, wt alpha-synuclein incubated with 10% of (a) wt alpha-synuclein, (b) P253, (c) P254, (d) P255, or (e) P256. alpha-synuclein fibrillation monitored by ThT fluorescence (ex450 / em480) every 10 min. Wt alpha-synuclein at 40 pM were incubated with 10% alpha-synuclein mutants and 0,1% PFF seeds in PBS at 37 °C with 5 min orbital shaking before each measurement. An average of three replicates is shown. Detailed ion

[0049] References to amino acid numbers herein refer to amino acids in SEQ ID NO 1. When the protein is truncated or otherwise mutated, the skilled person is able to identify the corresponding amino acids in the truncated or mutated protein by aligning the sequences, for example using Blast2sequences using default settings.

[0050] In some embodiments, parts of the C-terminal or N-terminal of the protein of SEQ ID NO 1 is removed to provide a truncated protein. For example, in some embodiments a protein corresponds to, comprises or consists of the amino acid residues 26-65 of SEQ ID NO lln some embodiments the protein has at least one mutation compared to amino acid sequence of residues 26-65 of SEQ ID NO 1 or a sequence that has at least 80 % identity with residues 26-65 of SEQ ID NO 1. In some embodiments the protein comprises or consists of the core hairpin sequence (amino acids 38-53 of SEQ ID NO 1) or a sequence that has a least 80 % identity to SEQ ID NO 38-53 of SEQ ID NO 1. In some embodiments, a truncated form of the protein may optionally have a suitable number of amino acids in the C-termi- nal or N-terminal, such as from or from 1 to 5, 1-10 or 1-20 or 1 to 37 additional amino acids in the C-terminal and / or N-terminal. The suitable number of amino acids in the C-ter- minal or N-terminal may be any suitable amino acids, such as a peptide tag, for example his tag or a flag tag. The truncated protein is able to form oligomers as described herein.

[0051] References to protein sequences herein may also, in various embodiments, refer to sequences that are substantially identical to the reference sequence. "Substantially identical" shall, for amino acid sequences, mean a sequence that has a percent identity which is at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, 97%, or 98% and most preferably at least 99% identical to the reference sequence. Sequence identity is determined using sequence alignment. Percent identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity, using BLAST for proteins (BLASTP) (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403-10. Doi: 10.1016 / S0022-2836(05)80360-2. PMID: 2231712.) BLAST is used with default settings (as of March 2023 which are: word size: 3, gap penalty existence: 11, gap penalty extension: 1).

[0052] The mutant protein may comprise a suitable peptide sequence for purification, identification or use in screening such as a flag tag, a his tag, GST or a fluorescent protein.

[0053] The protein may be produced by chemically synthesizing the peptide chain. The peptides can be synthesized by methods known in the art. The peptides can be obtained pure and in large quantities by means of organic synthesis, such as solid phase synthesis. Methods for peptide synthesis are well known in the art, for example using a peptide synthesis machine. Of course, the peptides may be ordered from a peptide synthesis company.

[0054] Alternatively, the protein is produced in a suitable expression system. Production of protein with the use of expression systems is well known in the art. In general, Current protocols in Molecular Biology (John Wiley & sons) provides guidance for polynucleotide handling and manipulation, and protein expression, purification and handling.

[0055] A suitable starting point for generation of protein as described herein may be a DNA sequence for human alpha-synuclein, for example SEQ ID NO 10. Another suitable starting point may be the DNA sequence for human alpha-synuclein, available as NCBI Reference Sequence: NM_000345.4 (https: / / www.ncbi.nlm.nih.gOv / nuccore / NM_000345.4).

[0056] Site directed mutagenesis may be carried out using any suitable method including PCR- based sit directed mutagenesis or CRISPR gene editing. Alternatively, the desired DNA encoding the mutant is synthesized using chemical synthesis. Suitable expression systems include eukaryotic cells such as CHO cells, insect cells or bacteria. Often, E. coli is the preferred expression system because of its ease of use. However, in some embodiments a eukaryotic expression system may be preferred, for example when disulfide bridges are desired.

[0057] Typically, the production of protein involves cloning of the coding sequence for the protein into a plasmid suitable for expression. The plasmid preferably has a promotor that drives expression. For expression in E. coli the T7 promotor may be useful. For expression in mammalian cells, the CMV promotor may be useful.

[0058] The plasmid is introduced into the cells with the use of well-known transfection protocols, and stable or transient expressing cells are generated. Suitable transfection techniques may be the use of heat-shock, electroporation or the use of liposomes, such as Lipofec- tamine® or virus-based methods. Clones stably expressing the protein may be selected, expanded and propagated.

[0059] The proteins may be expressed with a suitable tag for purification of the protein, such as poly-His tag.

[0060] Purification of protein may be carried out as is known in the art and may include steps such as: cell lysis, centrifugation, gel filtration, chromatography such as for example affinity chromatography and dialysis.

[0061] The mutant form of alpha-synuclein protein may have an enhanced ability to form oligomers in vitro. In various embodiments, the mutant form has at least one mutation compared to the wild type alpha-synuclein protein as defined by SEQ ID NO 1. The mutation may be for example an amino acid substitution or amino acid insertion. The mutation may for example be the substitution or insertion of a at least one proline or at least two cysteine residues. The mutation is preferably in the region corresponding to residues 26 to 65 of SEQ ID NO 1. Moreover, an insertion or substitution mutation may suitably be located in the region corresponding to residues 34 to 43 or 48 to 57, more preferably residues 34- 41 or residues 50-57 of SEQ ID NO 1. In certain embodiments there is at least one insertion or substitution in each of these regions.

[0062] Oligomers are preferably soluble while fibrils are not soluble. Oligomers may be separated from fibrils by centrifugation 1 hour at 20 000 g. Oligomers will then be present in the supernatant whereas fibrils will sediment.

[0063] Without being bound by any theory, it is believed that the mutations described stabilize a hairpin structure located at residues 38-53 of SEQ ID NO 1. The stabilized hairpin structure is believed to promote the formation of oligomers in the mutant alpha-synuclein, but prevent the mutant alpha-synuclein from converting into fibrils. The hypothetical hairpin structure has a beta turn involving residues 44-47 of SEQ ID NO 1. The center of the turn is at the bond between residues 45 and 46. Hence in various embodiments, the mutation stabilizes a hairpin structure in the region corresponding to amino acids 38 to 53 in SEQ ID NO 1. In various embodiments, the hairpin structure comprises a beta turn involving amino acid residues corresponding to residues 44-47 in SEQ ID NO 1.

[0064] The mutant synuclein protein has an enhanced ability to form oligomers, preferably in vitro. The oligomers may for example be dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, 10-mers, 20-mers, 30-mers or 50-mers. The oligomer may consist of any number of protein subunits from 2-100 or from 9 to 50 (such as, for example 17-mers or 31-mers). In various embodiments the oligomers are formed by from 2 to 100 subunits, from 2 to 60 subunits, from 2 to 50 subunits, from 2 to 40 subunits, from 2 to 30 subunits, from 2 to 30 subunits, from 2 to 20 subunits or from 2 to 10, 11, 12, 13, 14, 15, 16, 18 or 19 subunits. The ability to form oligomers is increased with at least 20 % more preferably with at least 50%, more preferably at least 75%, 100%, 200%, or 300 % compared to the wild-type protein as defined by SEQ ID NO 1. Hence an oligomer may comprise or consist of at least two peptide chains as described herein. Various mutants may have preference or a certain type of oligomer. For example, a first mutant may predominantly form tetramers, whereas a different form may predominantly form 10-mers. Examples can be seen in Fig 3.

[0065] The percentage increase is given for a single type of oligomer. Hence for example, the ability for forming trimers is increased with at least 75 %, or the ability to form hexamers is increased with at least 200%

[0066] Various mutants may be able to form one certain type of oligomer to a greater extent than the wild type protein.

[0067] The extent of oligomer formation is determined as the molarity of a certain oligomer. Any suitable method can be used, but in particular size exclusion chromatography may be used to determine the molarity of an oligomer in a solution.

[0068] Specifically, oligomer formation may be determined by allowing oligomers to form under the following conditions: PBS pH 7.4, with 0.1 % (by molarity relative to molarity of alpha- synuclein monomers) aggregation seeds (Polinski, N.K., et al., 2018. JPD 8, 303-322. https: / / doi.org / 10.3233 / JPD-171248), shaking at 37°C for 125 hours (Buell, A.K., et al., 2014. Proc. Natl. Acad. Sci. U.S.A. Ill, 7671-7676. https: / / doi.org / 10.1073 / pnas.1315346111; Doherty, C.P.A., et al., 2020. Nat Struct Mol Biol 27, 249-259. https: / / doi.org / 10.1038 / s41594-020-0384-x). Any potentially formed fibrils are removed by centrifugation 1 hour at 20000 g and oligomer formation is determined by size exclusion chromatography and compared to oligomerization of wild type protein (SEQ ID NO 1). The increase in formation of a certain oligomer is determined as:

[0069] % increase = (mutant oligomer molarity - wt oligomer molarity) / wt oligomer molarity.

[0070] A suitable resin for size exclusion chromatography may have a separation range of 10-600 kDa, for example Superdex™ 200 Increase. In various embodiments, fibril formation of the mutant protein is inhibited with at least 50%, more preferably 75 % even more preferably 90 %, compared to the wild type protein as determined by the method described in the Examples. Inhibition of fibril formation is determined as:

[0071] % decrease = (wt fibril molarity - mutant fibril molarity) / wt fibril molarity.

[0072] Particular suitable mutations include the substitution or insertion of a proline residue in an amino acid position that corresponds to residues 26-37 of SEQ ID NO 1, in particular positions 31-36 of SEQ ID NO 1, or positions 31-34 and 36 SEQ ID NO 1, or the substitution or insertion of a proline residue in an amino acid in an amino acid position that corresponds to residues 54-65 of SEQ ID NO l.lt may be suitable that these embodiments are combined hence that there is a first proline in an amino acid position that corresponds to residues 26-37 of SEQ ID NO 1, and second proline residue in an amino acid in an amino acid position that corresponds to residues 54-65 of SEQ ID NO 1. In an even more preferred embodiment, the first proline is a position that corresponds to residues 34-36 of SEQ ID NO 1 and the second proline is a position that corresponds to residues 56 or 57 of SEQ ID NO 1.

[0073] Particularly well suited mutations include a first proline substitution in one of the positions corresponding to V26, A27, E28, A29, A30, G31, K32, T33, K34, E35 or G36, V37 of SEQ ID NO 1 and a second proline substitution is in one of the positions corresponding to T54, V55, A56, E57, K58, T59, K60, E61, Q62, V63, T64, N65 in SEQ ID NO 1.

[0074] Without being bound by any theory, the prolines may disturb secondary structures outside the hairpin loop, causing the hairpin to form to a greater extent. Without being bound by any theory a proline may be suitable because it has an uncharged side chain.

[0075] In various embodiments the first and second proline residues are at the same distance from a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1, or where the two distances differ with at most two residues, preferably at most one residue, from a position corresponding to the bond between a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1.

[0076] A second set of suitable mutations is the substitution or insertion of a first cysteine residue in a first amino acid position that corresponds to positions 35 to 43, more preferably 35-41 and even more preferably 36-41 in SEQ ID NO 1 and the substitution or insertion of a second cysteine residue in a second amino acid position that corresponds to positions 48 to 56, more preferably 50-56 and even more preferably 50-55 in SEQ ID NO 1, where the first and second cysteine residues are at the same distance, from a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1, or where the two distances differs with at most two residues from a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1.

[0077] Without being bound by any theory it is believed that this enables the formation of a disulfide bridge between the two cysteine residues, which stabilizes the hairpin. Hence it may be useful to use oxidizing conditions in order to allow the formation of disulfide bridges.

[0078] Suitable sites for cysteines are the following pairs of position, or corresponding sequences, in SEQ ID NO 1: E35 / A56 or G36 / V55 or V37 / T54 or L38 / A53 or Y39 / V52 or V40 / G51 or G41 / H50 or S42 / V49 or K43 / V48 or E35 / T54 or G36 / A53 or V37 / V52 or L38 / G51 or Y39 / H50 or V40 / V49 or G41 / V48 or V37 / A56 or L38 / V55 or Y39 / T54 or V40 / A53 or G41 / V52 or S42 / G51 or K43 / H50.

[0079] Hence in various embodiments the mutation is a pair of cysteine substitutions in one of these pairs of positions.

[0080] In various embodiments the proline mutations and the cysteine mutations are combined such that one or two prolines are used and two cystines are used. For example, the following combinations of mutations may be suitable:

[0081] Table 1

[0082] SEQ ID NO 2, 3, 4 and 5 are examples only and the mutation of Table 1 may be used in positions corresponding to those positions. Non-limiting examples of coding DNA sequences are also provided in Table 1.

[0083] In various embodiments, the protein is at least partly in oligomeric form. The oligomeric form may be in water such as a water-based solution. The extent of oligomerisation may depend on the conditions and the mutants. In various embodiments 1-100 % of the protein is in any oligomeric form, more preferably 10 % - 90 % even more preferably from 20% to 70 %, but not fibrils. In various embodiments at least 10%, 20 %, 30%, 50%, 60%, 80%, 90 % of the protein is in any oligomeric form.

[0084] In some embodiments the oligomers are provided in freeze-dried form.

[0085] Oligomers can be formed using soluble protein (monomers) as the starting material in any suitable conditions. Suitable buffers include PBS, Tris, HEPA or MES buffer. The buffer has suitable pH which may be from 3 to 9, more preferably from 4 to 8. Any suitable temperature such as from +2C to +60C may be used, but it may be suitable to use a physiological temperature such as +37C. It may be useful to introduce aggregation initiators such as 0.1 % by molarity of alpha-synuclein aggregation seeds (Buell, A.K., et al., 2014. Proc. Natl. Acad. Sci. U.S.A. Ill, 7671-7676. https: / / doi.org / 10.1073 / pnas.1315346111; Polinski, N.K., et al., 2018. JPD 8, 303-322. https: / / doi.org / 10.3233 / JPD-171248). Oligomer formation may occur during agitation, such as during using a horizontal shaker (Giehm, L., Otzen, D.E., 2010. Anal Biochem 400, 270-281. https: / / doi.Org / 10.1016 / j.ab.2010.02.001). Agitation may be carried out during a time period of from 0.5 hours to 125 hours. Any potentially formed fibrils are preferably removed by centrifugation for example 1 hour at 20 000g.

[0086] The mutant synuclein protein, in particular oligomers, may be used to determine the binding of a molecule such as a synuclein binding agent towards the protein. This may be used to identify drug candidates for the treatment of Alzheimer's. For example, the affinity of a molecule towards the mutant synuclein protein or oligomer may be determined. The binding of the candidate molecule toward the mutant protein may be compared to the binding of the candidate molecule to the wild type protein (SEQ ID NO 1), preferably in vitro. It is desirable that drug candidates have a higher binding toward the mutant protein than the wild-type protein. In various embodiments the molecule is a protein, a peptide or a small molecule. In preferred embodiments the molecule is a protein or a peptide, in particular a protein.

[0087] Oligomers may be used in screening, for example in screening of molecules that are drug candidates. Binding of a candidate binding agent (molecule) may be determined by methods known in the art. For example, a candidate molecule, such as an antibody or antibody fragment is allowed to bind to the oligomers and the affinity is determined. Suitable technologies include phage display, ELISA, Surface plasmon resonance, LigandTracer, and mass photometry. It is preferred that the candidate binder is immobilized, and that the oligomer is in solution. However, other conformations may be used, for example the oligomer may be immobilized, and the candidate molecule may be in solution. In some embodiments a library of candidate binding agents is screened for binding. Suitable reporter systems may be used including labelled candidate drugs, such as for example labelling with enzymes or fluorescence. Examples of useful protocols include those described in: Rohit Singh, Pankaj Chandley, Soma Rohatgi, Recent Advances in the Development of Monoclonal Antibodies and Next-Generation Antibodies, ImmunoHorizons, Volume 7, Issue 12, December 2023, Pages 886- 897, https : / / doi . org / 10 . 404 9 / immunohori z ons . 2300102, Parray HA, Shukla S, Samal S, Shrivastava T, Ahmed S, Sharma C, Kumar R. Hybridoma technology a versatile method for isolation of monoclonal antibodies, its applicability across species, limitations, advancement and future perspectives. Int Immunopharmacol. 2020 Aug;85:106639. doi: 10.1016 / j.intimp.2020.106639. Epub 2020 May 27. PMID: 32473573; PMCID: PMC7255167; Schirrmann T, Meyer T, Schutte M, Frenzel A, Hust M. Phage display for the generation of antibodies for proteome research, diagnostics and therapy. Molecules. 2011 Jan 10;16(l):412-26. doi: 10.3390 / moleculesl6010412. PMID: 21221060; PMCID: PMC6259421; Pedrioli A, Oxenius A. Single B cell technologies for monoclonal antibody discovery. Trends Immunol. 2021 Dec;42(12):1143-1158. doi: 10.1016 / j.it.2021.10.008. Epub 2021 Nov 4. PMID: 34743921.

[0088] EXAMPLES

[0089] Example 1

[0090] The wildtype human alpha-synuclein protein (SEQ ID NO 1) were expressed in E. coli. In addition, mutants P253 (SEQ ID NO 2), P254 (SEQ ID NO 3), P255 (SEQ ID NO 4) and P256 (SEQ ID NO 5) were expressed in E. coli. Fig. 1 is an overview of the mutations of the mutant proteins.

[0091] Example 2

[0092] The proteins were allowed to aggregate as described in the methods section, below. The results are shown in Fig. 2. The increase of the ThT fluorescence signal in (a) indicated the formation of wt alpha-synuclein fibrils as ThT molecules intercalate in the cross-beta-sheet structure of fibrils. The lack of ThT fluorescence increase in (b), (c) and (e) indicate the absence of fibrils or a disturbed ThT-binding, thus P253, P254 and P256 likely do not form any fibrils. The subtle increase of ThT fluorescence in (d) indicates that P255 forms fibrils but less than wt alpha-synuclein or fibrils to which ThT has a lower affinity compared to wt alpha-synuclein fibrils. Example 3

[0093] The molecular mass of soluble alpha-synuclein species after incubation under aggregation conditions was determined by mass photometry and the results are shown in Fig. 3 and Table 2.

[0094] Table 2: Mass distribution of soluble species after seeded aggregation measured by mass photometry.

[0095] The mass distribution analysis in Fig. 3 and Table 2 shows the molecule mass of soluble species present in the supernatant fraction of wt alpha-synuclein or alpha-synuclein mutants after incubation under aggregation conditions as shown in Fig. 2. The results indicate that there was not a large variation in molecule masses, instead each protein formed oligomers of a distinct molecule mass. These results indicate that P253 forms predominantly hexamers, P254 pentamers, and P255 and P256 a mixture of tetramers and pentamers. Oligomers of tetrameric size were found for wt alpha-synuclein but at a lower molecule count which aligns with the observation in Fig. 2 that wt alpha-synuclein forms predominantly insoluble fibrils which were removed from the supernatants prior to mass photometry analysis.

[0096] Example 4

[0097] Circular dichroism (CD) spectra between 190 and 260 nm were recorded for the aggregates.

[0098] The CD analysis showed that all proteins have a predominantly disordered structure in their monomeric form Fig. 4 (a) which can be seen by the negative peak at 200 nm wavelength. The lower amplitude of this peak in the alpha-synuclein mutant samples compared to the wt alpha-synuclein sample shows that all alpha-synuclein mutants contain a higher degree of secondary structure than wt alpha-synuclein. This gain of secondary structure is likely due to the presence of the beta-hairpin in the mutants.

[0099] Conclusions Fig. 4 b: After incubation under aggregation conditions like in Fig. 2, wt alpha- synuclein adopted a predominant beta-sheet structure, which can be seen by the slightly positive peak at 200 nm and the negative peak at 220 nm, and which aligns with the observation in Fig. 2 that wt alpha-synuclein forms fibrils.

[0100] Example 5

[0101] The co-incubation of wt alpha-synuclein with 10% alpha-synuclein mutants shows a delayed increase in ThT fluorescence and a lower plateau of the total ThT signal compared to when wt alpha-synuclein is incubated alone (Fig 5). These results indicate that the presence of 10% alpha-synuclein mutants delay the fibrillation and decrease the fibril yield.

[0102] Methods in

[0103] Lyophilized wt alpha-synuclein or mutant alpha-synuclein was dissolved in ice-cold, sterile PBS (Gibco 14190-144) to a concentration of 2 mg / ml. To remove any preformed aggregates, the protein solution was filtered through a 100 K MWCO centrifugal filter (Millipore UFC510024) at 14.000 x g, 4°C. The protein concentration was determined by the protein's absorbance at 280 nm measured on a DS-11 spectrophotometer (DeNovix, Wilmington, USA) together with the protein's molecular weight and extinction coefficient, which were calculated from the protein sequence using the ProtParam tool on the ExPASy server (Gas- teiger, E., 2003. Nucleic Acids Res 31, 3784-3788. https: / / doi.org / 10.1093 / nar / gkg563). fibrils (PFF) seeds

[0104] Lyophilized wt alpha-synuclein (Alexotech, AS-600-100) was dissolved in ice-cold, sterile PBS to a concentration of 3 mg / ml and incubated in a sterile 1,5 ml Eppendorf (Sarstedt 72.690.001) tube at 37 °C with constant shaking at 600 rpm in a Thermoshaker (Thermal Shake lite, VWR, Leuven, Belgium) for 7 days. The fibrils were harvested by centrifugation at 21.000 x g for 30 min, the pellet was resuspended in a small volume of PBS, aliquoted and stored at -80°C. To determine the protein concentration in the fibril fraction, insoluble aggregates were dissolved in 1% SDS before measuring the protein's absorbance at 280 nm measured on a DS-11 spectrophotometer (DeNovix, Wilmington, USA). PFF aliquots were thawed at RT, diluted 1:10 in PBS and sonicated for 10 min in a Branson 5510 bath sonicator (Branson Ultrasonics, Brookfield, USA) just before being used as seeds in the aggregation assay.

[0105] Aggregation assays were performed in non-binding, black with clear bottom, half-area 96- well plates (Corning 3881). Wt alpha-synuclein or alpha-synuclein mutants at 40 pM were prepared in ice-cold PBS with 10 pM ThT (Sigma T3516). For seeded aggregation, 0,1% sonicated PFF (40 nM, in monomer equivalents) were added to the reaction mix. For the co-incubation aggregation assay, 10% (4 pM) of the respective monomeric alpha-synuclein mutant and 0,1% sonicated PFF were added to 40 pM wt alpha-synuclein. A glass bead of 2 mm diameter (Lenz 05124002) was added to each reaction well to promote fibril fragmentation and to ensure homogeneous mixing during the incubation (Buell et al., 2014; Cohen et al., 2012). The plate was sealed with clear adhesive PCR film (Thermo scientific AB-0558) and placed in an Infinite M Nano+ plate reader (Tecan, Mannedorf, Switzerland) which was prewarmed to 37 °C. The ThT signal intensity was measured every 10 min with excitation at 450 nm (9 nm bandwidth), emission at 480 nm (20 nm bandwidth), 6 flashes per reading and a gain of 60. Both the seeded and the co-incubation aggregation assay were incubated with 5 min orbital shaking (amplitude 1 mm, frequency 432 rpm) before each measurement. Mass photometry

[0106] The molecular mass of soluble alpha-synuclein species after incubation under aggregation conditions was determined by mass photometry on a Refeyn 2MP instrument (Refeyn

[0107] 5 Ltd., Oxford, UK). The instrument was calibrated with NativeMark Unstained Protein Standard (Thermo Scientific LC0725). Aggregation of alpha-synuclein wt and mutants was carried out as described above and supernatants were harvested after 64 h aggregation by centrifugation in low-binding tubes (Eppendorf 525-0133) at 15.000 x g for 1 h at 37 °C. Supernatants were kept at RT and were diluted 1:200 in PBS directly before the measure- w ment.

[0108] Circular dichroism (CD)

[0109] CD spectra between 190 and 260 nm were recorded on a JASCO J-1500 CD spectrometer 15 (JASCO, Easton, MD, USA) at 25 °C using quartz cuvettes (Hellma Analytics 110-1-40) with a path length of 1 mm. The samples taken at the end of ThT aggregation assays were diluted to a protein concentration of 0,2 mg / ml in PBS and measured at a scanning speed of 50 nm / min with a step size of 0,1 nm. Spectra of three scans per sample were averaged and baseline-corrected by subtracting the buffer spectrum. 0

Claims

23CLAIMS1. A mutant alpha synuclein protein where the amino acid sequence of mutant alpha synuclein has at least 80 % identity to SEQ ID NO 1, or at least 80% identity to a truncated version of SEQ ID NO 1 corresponding to at least residues 26 to 65 of SEQ ID NO 1, where the mutation is at least one of a) the substitution or insertion of a proline residue in an amino acid position that corresponds to residues 26-37 of SEQ ID NO 1, or b) the substitution or insertion of a proline residue in an amino acid in an amino acid position that corresponds to residues 54-65 of SEQ ID NO 1, or c) the substitution or insertion of a first cysteine residues in a first amino acid position that corresponds to positions 35 to 43 in SEQ ID NO 1 and the substitution or insertion of a second cysteine residue in a second amino acid position that corresponds to positions 48 to 56 in SEQ ID NO 1, where the first and second cysteine residues are at the same distance from a position corresponding to the bond between a position corresponding to residues 45 and 46 in SEQ ID NO 1, or where distances from each of the cysteines to a position corresponding to the bond between residues 45 and 46 in SEQ ID NO 1 differs with at most two residues.

2. The mutant alpha-synuclein according to claim 1 where the mutations comprise a) and b) or a) and b) and c) or a) and c) or b) and c).

3. The mutant synuclein protein according to any one of claims 1 to 2 wherein the molarity of an oligomer selected from a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, 10-mer, 20-mer, 30-mer or 50-mer of the alpha-synu- clein protein is enriched in vitro with at least 50% compared to the wild-type protein as defined by SEQ ID NO 1.

4. The mutant synuclein protein according to claim 3 where the molarity of the oligomer is determined by size exclusion chromatography.

5. The mutant synuclein protein according to any one of claims 1 to 4 where fibril formation of the mutant protein is inhibited with at least 50 % compared to the wild type protein as defined by SEQ ID NO 1.

6. The mutant alpha-synuclein according to any one of claims 1 to 5 where the mutation is at leastG36P and A56P,G36P, V37C, T54C and A56P,K34P, E35C, A56C and E57P, orG36P, G41C, H50C and A56P, where the amino acid position corresponds to the amino acid positions in SEQ ID NO 1.7 . The mutant synuclein protein according to any one of claims 1 to 6 comprising one of SEQ ID NO 2 to SEQ ID NO 5.8 . The mutant synuclein protein according to any one of claims 1 to 7 in oligomer form.9 . A polynucleotide that codes for the protein according to any one of claims 1 to 8.

10. A method for determining binding of a molecule to a pathogenic form of synuclein comprising contacting the molecule with a mutant synuclein protein according to any of claims 1 to 8 in vitro and determining binding of the molecule to the synuclein protein.

11. The method of claim 10 where the mutant synuclein protein is in oligomer form.

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