Method of identifying novel protein aggregation inhibitors based on chemical kinetics

#This application is a U.S. National Stage entry of International Application No. PCT/GB2016/054083 filed Dec. 28, 2016, which claims the benefit of United Kingdom Patent Application No. 1600176.0, filed Jan. 6, 2016, each of which is herein incorporated by reference in its entirety for all purposes.

#The present invention relates to methods based on chemical kinetics for identifying novel inhibitors of protein aggregation, particularly inhibitors that inhibit aggregation of amyloid proteins associated with human misfolding diseases. The invention also relates to a method of designing and generating a pharmacophore against protein aggregation. Also within the scope of the invention are compositions and methods for the treatment of Alzheimer's disease and for the inhibition of protein aggregation in other protein misfolding diseases.

#Intracellular protein misfolding or aggregation is a feature of many late-onset neurodegenerative diseases, called proteinopathies. These include Alzheimer's disease, Parkinson's disease, tauopathies, and polyglutamine expansion diseases, such as Huntington's disease and various spinocerebellar ataxias (SCAs), like SCA3.

#FIG. 1: Schematic illustration of the drug discovery strategy described in this work. The strategy consists of 4 steps: (1) A fragment-based approach that allows the identification of small molecules that interact with the aggregation-prone system, here Aβ42, including FDA approved molecules for drug repurposing; (2) An in vitro kinetic analysis that identifies the specific molecular steps in the Aβ42 aggregation mechanism responsible for the generation of toxic species; (3) A further kinetic analysis to determine the mechanism of inhibition associated with the molecules identified in (1); and (4) An evaluation of the effects of these molecules on the formation of toxic species in vivo. In particular, the inhibition of primary nucleation is predicted only to delay the aggregation without affecting the total number of oligomers generated by the aggregation process, while inhibiting elongation or secondary nucleation is predicted either to increase or to decrease the number of toxic oligomers, respectively.

#FIG. 2: Bexarotene, but not tramiprosate, delays the formation of Aβ42 fibril formation. (a) Kinetic profiles of Aβ42 aggregation under quiescent conditions at a concentration of 3 μM (open circles) and 4 μM (open squares) in the absence or in the presence of a 4-fold excess of tramiprosate. (b) Kinetic profiles of Aβ42 aggregation under quiescent conditions at a concentration of 3 μM (open circles) and 4 μM (open squares) in the absence or in the presence of a 4-fold excess of bexarotene. (c) Average half-time of the aggregation reaction as a function of the initial monomer concentration in the absence or in the presence of 4-fold excess of bexarotene.

#FIG. 3: Bexarotene delays Aβ42 fibril formation in a label-free environment. (a) Kinetic profiles of the aggregation of 2 μM Aβ42 under quiescent conditions in the absence and in the presence of a 4-fold excess of bexarotene; the table below the graph shows the equivalent of the different time points in hours (represented in black solid lines in the graph) at which aliquots of Aβ42 were removed from a solution of 2 μM peptide undergoing aggregation. (b) AFM images of Aβ42 species in the absence and in the presence of a 4-fold excess of bexarotene. Images were acquired with tapping mode in air on aliquots of the Aβ42 solutions that were removed from the aggregation reaction at 1.2 and 2.1 h time points. Note that fibrillar structures can be observed after 2.1 h only in the absence of bexarotene. (c) Time course of the formation of 2 μM Aβ42 fibrils as assessed by antibody binding. The quantity of Aβ42 peptide that was detected by the sequence-specific W0-2 antibody (upper panel) remained unchanged during the complete time course of the reaction on the total quantity of Aβ42 (in solution or as aggregates). The fibril-specific OC antibody (lower panel), however, probes only fibrillar structures that can be seen to have formed earlier in the absence of bexarotene than in its presence. The extent of the observed delay (highlighted in red) is in complete accord with the aggregation profiles shown in (a). (d) Calibration of the dependence of Aβ42 fibril mass concentration to the dot-blot intensity of the fibril-specific OC antibody. (Top) Kinetic profile of 4 μM Aβ42 by means of ThT fluorescence. AFM image of typical mature Aβ42 fibrils acquired with tapping mode in air, formed at pH 8. (Bottom) Dot-blot intensities obtained from binding of fibril-specific OC antibody to a serial dilution of Aβ42 fibrils that were collected after 3 hours incubation of a fresh 4 μM Aβ42 monomer. Fibril concentrations were in the range of 4 μM to 0.1 μM.

#The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

#The invention relates to a method for the identification of novel compounds that can act to inhibit aggregation of aggregation-prone proteins such as amyloid proteins. In particular, the invention relates to a method of identifying a compound that can inhibit one or more of the specific microscopic events, in particular primary and secondary nucleation, that underlie the process of protein aggregation.

#As discussed in Arosio et al. (2014) (28) incorporated herein by reference, protein aggregation can be considered a multi-step process beginning with a nucleation step that generates small oligomers from a pool of monomeric proteins. Such oligomers are very reactive and metastable, and consequently will rapidly elongate into protofibrils and then fibrils by monomer addition. In most cases however, the aggregation rate is then exponentially accelerated by secondary nucleation processes, such as fibril fragmentation and surface catalysed nucleation, which form new reactive nuclei and result in a high level of apparent cooperativity in the nucleation process.