Systematic Development of Small Molecules to Inhibit Specific Microscopic Steps of Aβ42 Aggregation in Alzheimer’S Disease

Systematic development of small molecules to inhibit specific microscopic steps of Aβ42 aggregation in Alzheimer’s disease

Johnny Habchia,1, Sean Chiaa,1, Ryan Limbockera, Benedetta Manninia, Minkoo Ahna, Michele Pernia, Oskar Hanssonb,c, Paolo Arosioa, Janet R. Kumitaa, Pavan Kumar Challaa, Samuel I. A. Cohena, Sara Linsed, Christopher M. Dobsona,2, Tuomas P. J. Knowlesa,2, and Michele Vendruscoloa,2

aDepartment of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom; bDepartment of Clinical Sciences, Lund University, 221 00 Lund, Sweden; cMemory Clinic, Skåne University Hospital, 205 02 Malmo, Sweden; and dDepartment of Biochemistry & Structural Biology, Center for Molecular Protein Science, Lund University, 221 00 Lund, Sweden

Edited by William A. Eaton, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, and approved November 23, 2016 (received for review September 18, 2016) The aggregation of the 42-residue form of the amyloid-β peptide delays primary nucleation in Aβ42 aggregation (22) (Fig. 1A). (Aβ42) is a pivotal event in Alzheimer’sdisease(AD).Theuseof Because primary nuclei form only transiently during the aggrega- chemical kinetics has recently enabled highly accurate quantifications tion process (21, 23, 24), it is extremely challenging to characterize of the effects of small molecules on specific microscopic steps in Aβ42 their structures experimentally, making it difficult to apply to them aggregation. Here, we exploit this approach to develop a rational structure-based drug discovery strategies. The structural features of drug discovery strategy against Aβ42 aggregation that uses as a read- these transient nuclei, however, may be shared with other biolog- out the changes in the nucleation and elongation rate constants ical targets of bexarotene, which was initially identified as a retinoid caused by candidate small molecules. We thus identify a pool of X receptor (RXR) agonist and approved by the US Food and Drug compounds that target specific microscopic steps in Aβ42 aggrega- Administration for the treatment of cutaneous T-cell lymphoma tion. We then test further these small molecules in human cerebro- (26). Ligands that bind RXRs and their partners, the retinoid A spinal fluid and in a Caenorhabditis elegans model of AD. Our results receptors (RARs), modulate the communication of these recep- show that this strategy represents a powerful approach to identify tors with their intracellular environments (27, 28). The mecha- systematically small molecule lead compounds, thus offering an ap- nisms of binding of the ligands to the RAR and RXR ligand- pealing opportunity to reduce the attrition problem in drug discovery. binding domains (LBDs) are well understood and are exploited for pharmaceutical purposes (27, 29). We took advantage of the data Alzheimer’s disease | amyloid-β peptide | protein misfolding | drug available on the atomic structures of the LBDs and the chemical discovery | protein aggregation properties of the known agonists and antagonists of RARs and

lzheimer’s disease (AD) is, to date, an incurable neurode- Significance Agenerative disorder that imposes substantial social and eco- nomic costs worldwide (1). According to the amyloid hypothesis, The absence of fully reproducible protein aggregation assays the aggregation of the amyloid-β peptide (Aβ) initiates a cascade of has contributed to the systematic failures in clinical trials for molecular events leading eventually to neuronal death (2–11). Be- Alzheimer’s disease (AD) of compounds targeting the aggre- cause the presence of abnormal Aβ metabolism can be detected 10– gation process of the amyloid-β peptide (Aβ). To address this 20 years before the onset of AD (12, 13), early interventions may be problem, we report the identification of a library of com- possible before widespread and irreversible neurodegeneration has pounds against Aβ aggregation using a drug discovery strategy occurred. Although targeting Aβ accumulation has been pursued as based on highly quantitative aggregation rate measurements. a major potential therapeutic strategy against AD (14–17), no com- We then demonstrate, both in Caenorhabditis elegans and pound selected for this purpose has yet entered clinical use (18, 19). human cerebrospinal fluid, that this approach can systemati- Although these failures have raised doubts about the amyloid hy- cally provide a rich variety of related small molecules to take pothesis (20), they can also be attributed to an incomplete knowledge forward into a drug discovery process. We therefore report an of the molecular mechanisms by which the compounds tested so far approach that should substantially help overcome the very high affect the nucleation and growth of Aβ aggregates. Indeed, it has been level of attrition associated with drug discovery programs for AD. shown that inhibiting Aβ aggregation without a detailed understanding of the underlying microscopic processes could affect the toxicity in Author contributions: J.H., S.C., R.L., M.P., P.A., S.L., C.M.D., T.P.J.K., and M.V. designed unexpected ways (21, 22). For example, the inhibition of nucleation research; J.H., S.C., R.L., B.M., M.A., M.P., O.H., P.A., J.R.K., P.K.C., and S.L. performed research; J.H., S.C., R.L., B.M., M.A., M.P., O.H., P.A., J.R.K., P.K.C., S.L., C.M.D., T.P.J.K., events may delay or decrease toxicity, whereas the inhibition of elon- and M.V. contributed new reagents/analytic tools; J.H., S.C., R.L., B.M., M.A., M.P., O.H., gation may lead to an overall increase in toxicity (21, 22). Therefore, P.A., J.R.K., P.K.C., S.I.A.C., S.L., C.M.D., T.P.J.K., and M.V. analyzed data; and J.H., S.C., R.L., effective therapeutic strategies must be aimed at targeting precise B.M., M.A., M.P., O.H., P.A., J.R.K., P.K.C., S.I.A.C., S.L., C.M.D., T.P.J.K., and M.V. wrote the paper. microscopic steps during the Aβ aggregation process (21, 23–25). Conflict of interest statement: Part of the work described in this paper has been the We describe here the development of a systematic pipeline subject of a patent application filed by Cambridge Enterprise, a wholly owned subsidiary of the University of Cambridge (now licensed to Wren Therapeutics Ltd., where M.V. is based on chemical kinetics to identify a pool of candidate mol- Chief Scientific Officer; S.I.A.C., C.M.D., and M.V. are members of the Board of Directors; ecules directed against the aggregation of the 42-residue form and S.I.A.C., S.L., C.M.D., and T.P.J.K. are consultants). of Aβ (Aβ42), and to understand the key chemical features re- This article is a PNAS Direct Submission. sponsible for their inhibitory activity. Freely available online through the PNAS open access option. 1J.H. and S.C. contributed equally to this work. Results and Discussion 2To whom correspondence may be addressed. Email: [email protected], [email protected]. A Quasi-Structure–Based Drug Discovery Strategy. We introduce first a uk, or [email protected]. – quasi-structure based drug discovery (QSBDD) strategy, which This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. builds on the recent finding that the small molecule bexarotene 1073/pnas.1615613114/-/DCSupplemental.

E200–E208 | PNAS | Published online December 23, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1615613114 Downloaded by guest on September 24, 2021 RXRs, and applied QSBDD by presuming that, like bexarotene, of an inhibitor are described by introducing into the rate laws PNAS PLUS other RXR and RAR ligands may inhibit the aggregation of Aβ42. suitable perturbations to each of the microscopic rate constants We thus collected a group of 12 small molecules (Fig. 1B), in- evaluated in the absence of the inhibitor. The modifications of cluding five RAR agonists (tamibarotene, BMS753, adapalene, the rate constants required to describe the aggregation profiles CD1530, TTNPB, and Ch55), four RAR antagonists (BMS195614, in the presence of different concentrations of inhibitor are then LE135, MM11253, and BMS493), two RXR agonists (bexarotene indicative of the specific process affected by the presence of the and SR11237), and one RXR antagonist (UVI3003). We tested compound (21). these compounds in a thioflavin-T (ThT)-based chemical kinetics At low concentrations of small molecules, the data are ex- assay and compared their effects on the different microscopic steps tremely well described when the primary pathways [as expressed in the Aβ42 aggregation reaction (Fig. 1C). by the product of the of primary nucleation and rate of elongation (knk+)] are specifically decreased at less than a 3 M eq of set RAR and RXR Ligands Inhibit Aβ42 Aggregation to Different Extents. A and less than 0.5–1 M eq of set B molecules, where k+ is the β μ β We monitored A 42 fibril formation in vitro for 2 MA 42 in rate constant of fibril elongation and kn is the rate constant for the absence and presence of these small molecules. For Aβ42 primary nucleation (Fig. 3 A–C and F–H and Figs. S4 and S5). By alone the half-time of aggregation was about 2 h under the buffer contrast, at higher concentrations of small molecules, the data conditions used. For each compound except BMS753, we ob- are consistent with a decrease in the rate constants of both pri- β served substantial delays in A 42 aggregation when the com- mary (knk+) and secondary (k2k+) pathways, where k2 is the rate pounds were included at a concentration of 6 μM [3 molar constant of surface-catalyzed secondary nucleation. All kinetic equivalents (M eq)] (Fig. 2A). In most cases, the delays were curves were then compared with simulations where both primary greater than those assessed for bexarotene, and five of the 12 and secondary pathways were decreased concomitantly, and the molecules (MM11253, BMS493, adapalene, CD1530, and LE135) rates of both pathways were plotted against the concentration of inhibited the aggregation of Aβ42 completely over 10 h of ob- small molecules (Fig. 2 A–J and Figs. S4 and S5). This analysis servation (Fig. 2A). These five compounds were very effective in revealed that set A and set B molecules can affect both nucleation delaying the aggregation of Aβ42 even at substoichiometric ratios pathways in Aβ42 aggregation (Fig. 2 A–J and Figs. S4 and S5). (0.5 M eq; Fig. 2B). To investigate these effects further and to To quantify the effects of the small molecules on Aβ42 ag- exclude possible interferences of the compounds with ThT binding gregation further, we examined the increase in ThT fluorescence to Aβ42 fibrils and the fluorescence measurements, we probed the at the end of the reaction, finding similar values in all cases (Figs.

quantities of Aβ42 fibrils at 12 time points during the aggregation S4H and S5F). These results suggest that a similar fibril mass NEUROSCIENCE reaction in the absence and presence of the small molecules using concentration is formed irrespective of whether or not the small a dot-blot assay with an Aβ42 fibril-specific antibody (OC; see molecules are present, in agreement with the dot-blot assays. In Methods)(Fig.2C and D and Fig. S1). The delay induced by the addition, the effect of the small molecules on the aggregation small molecules in the dot-blot assay was found to be identical kinetics of Aβ42 was found to be mainly determined by the molar within experimental error to the delay observed in the ThT- ratio of the small molecules with respect to Aβ42 rather than by based assay. its total absolute concentration (Fig. S6 A–C), thus implying that We subsequently classified these molecules according to their our experiments are conducted above the Kd for the relevant efficacy in inhibiting Aβ42 aggregation. The intensities of the dot interactions. Based on these results, given that the concentration blots (Fig. 2 C and D) were quantified and normalized against of Aβ42 is much lower in vivo than that used here in vitro, we the intensity of Aβ42 alone. The values at two early time points would expect that much lower concentrations of the compounds were plotted against the half-times derived from the ThT-based could be required to affect the rate constants of Aβ42 aggrega- kinetics (Fig. 2 E and F), resulting in a linear correlation, tion to the same extent, although the molar ratio might need to showing the high degree of consistency between the two assays. be increased in the concentration range below the value of Kd. This analysis allowed the classification of the RAR and RXR Furthermore, to rule out any possible interference of the small ligands in two sets according to the extent of the delay induced molecules on the aggregation pathway of Aβ42, such as a stabi- in Aβ42 aggregation (Fig. 2 E and F). Set A consists of seven lization of nonfibrillar aggregates, we used transmission electron molecules showing an effect at 3 M eq similar to or greater than microscopy (TEM) to image the Aβ42 species formed at the end the effect of bexarotene (Fig. 2E, light green), and set B consists of the aggregation reactions in the absence and presence of of five molecules that completely inhibited Aβ42 aggregation for two representative molecules from sets A and B, namely, at least 10 h at 3 M eq. We then analyzed the effects of these BMS195614 and LE135 (Fig. S6 E and F). In agreement with our molecules at substoichiometric concentrations by both assays previous atomic force microscopy findings on the effect of bex- (Fig. 2F, light orange). UVI3003 was identified as the most ef- arotene on the aggregation kinetics of Aβ42 (22), the TEM im- fective molecule within set A, delaying by more than fourfold the ages showed that similar Aβ42 fibrillar species are formed at the aggregation reaction, whereas MM11253 and adapalene were end of the aggregation reactions. found to be the most effective molecules within set B, because the aggregation of 2 μMAβ42 was delayed by at least threefold RAR and RXR Ligands Inhibit Secondary Pathways to Different Extents. at a concentration half of the concentration of Aβ42. Further- To explore further the effects of the molecules on distinct steps of more, RAR and RXR ligands inhibited Aβ42 reaction in a the secondary pathways further (i.e., on the surface-catalyzed concentration-dependent manner, with set B molecules showing secondary nucleation and elongation steps), we carried out an efficacy at concentration ratios as low as 0.2 (Fig. S2). The only additional series of kinetic measurements in the presence of the molecule that did not show any effect on Aβ42 aggregation was different compounds and various concentrations of preformed BMS753, even when present at 5 M eq (Fig. S3). fibril seeds. For about 10% of preformed fibrils, the primary and secondary nucleation steps are bypassed and the formation of RAR and RXR Ligands Inhibit Primary and Secondary Pathways. We mature fibrils is greatly accelerated by elongation reactions pro- next carried out a quantitative analysis of the effects of the moted by the fibril seeds (Fig. S7). Under these conditions, set A molecules by matching the experimental aggregation profiles to molecules did not affect the aggregation kinetics of 2 μMAβ42 kinetic curves obtained by using the rate laws derived from a even at a concentration of 5 M eq relative to the peptide (Fig. 4A), master equation that relates the time evolution of fibril forma- whereas the corresponding aggregation process under unseeded tion to the rate constants of the different microscopic events (21, conditions was slowed by at least a factor of 3 (Fig. 2A), strongly 24, 25). In this approach, the aggregation profiles in the presence indicating that the set A compounds have no effect on elongation.

Habchi et al. PNAS | Published online December 23, 2016 | E201 Downloaded by guest on September 24, 2021 A RAR RXR

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Fig. 1. Schematic illustration of the drug discovery strategy targeting Aβ42 aggregation described in this work. The strategy consists of two main steps: (i) a QSBDD step and (ii) a kinetics-based drug discovery (KBDD) step. QSBDD consists of identifying potential molecules against Aβ42 aggregation based on the structure of a receptor (here, RXR) of a small molecule (here, bexarotene) shown preferentially to inhibit a microscopic step in Aβ42 aggregation (here, primary nucleation). The rationale behind this strategy is that the instability and transient nature of Aβ42 oligomers make their characterization and, subsequently, the structure-based drug development very challenging. In this study, we considered known agonists and antagonists of RXR based on the possibility that structural similarities could exist between the binding pockets of RXR and Aβ42 oligomers. The strategy was extended to include agonists and antagonists of RAR, given the high structural similarities with RXR. In total, 13 molecules were tested, including bexarotene. (A) This panel is adapted from a study by Habchi et al. (22) and shows the kinetics of 5 μMAβ42 aggregation (black) in the presence of increasing concentrations of bexarotene (i.e., 1–5 M eq). The structure [Protein Data Bank (PDB) ID code 1XDK] of the RAR receptor (blue) is shown as a dimer with the RXR receptor (orange). The RXR subunit is superimposed onto the subunit of RXR with bexarotene in the binding pocket (green; PDB ID code 4K6I). An enlarged image of the binding pocket is also shown. (B) Structures of all the molecules tested in this study. (C) The KBDD step assesses the effect of a small molecule at the microscopic level. (C) A given molecule can bind monomers, primary oligomers, secondary oligomers, fibrils, or a combination of species, and accordingly affects different microscopic steps that can be characterized quantitatively by global fitting of the aggregation kinetics. The solid lines correspond to the aggregation kinetics of Aβ42 in the absence of an inhibitor. The dotted lines correspond to the kinetics of Aβ42 in the presence of inhibitors that affect a single microscopic step.

E202 | www.pnas.org/cgi/doi/10.1073/pnas.1615613114 Habchi et al. Downloaded by guest on September 24, 2021 PNAS PLUS AB 3 molar equivalents 0.5 molar equivalents

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Fig. 2. RAR and RXR ligands affect Aβ42 aggregation to different extents. (A) Kinetic profiles of the aggregation of a 2 μMsolutionofAβ42 in the absence and presence of 3 M eq of RAR and RXR ligands, shown in different colors. Note that Aβ42 did not aggregate within 10 h in the presence of five of the 13 molecules, seen as flat lines. (B) Kinetic profiles of 2 μMAβ42 aggregation in the absence and presence of substoichoimetricratiosofthefivemoleculesthatareshownasflatlinesinA.(C and D) Comparative time course of the formation of 2 μMAβ42 fibrils in the absence and presence of 3 M eq of the small molecules using a dot-blot assay. Aβ42 in (D, Top) did not aggregate in the presence of 3 M eq of adapalene, MM11253, BMS493, CD1530, and LE135; therefore, the aggregation kinetics of 2 μMAβ42 were repeated in the presence of substoichiometric quantities of the same molecules (0.5 M eq) (D, Bottom). The plots show, as histograms, the half-times of the aggregation reactions of Aβ42 from A and B and, as open circles, the correlation between the normalized dot-blot intensities with respect to the correlation in the presence of 1% DMSO at 1.6 h and2h(E)and1.5h and 2.5 h (F) of the aggregation reactions from C and D. The molecules were separated into two sets according to their potency, which was evaluated based on the extent of the delay that they induced in Aβ42 aggregation. (E) Set A (highlighted in light green) contains the molecules showing an effect similar to or greater than that of bexarotene (the half-time of Aβ42 aggregation in the absence and presence of bexarotene are highlighted with dotted black and red lines, respectively). AU, arbitrary units. (F) Molecules classified as set B (highlighted in light orange) inhibited the aggregation of Aβ42 completely within 10 h in the presence of 3 M eq of the small molecules.

Habchi et al. PNAS | Published online December 23, 2016 | E203 Downloaded by guest on September 24, 2021

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Fig. 3. Characterization of the effects of set A and set B molecules on Aβ42 aggregation using quantitative chemical kinetics. (A–C) Kinetic profiles of the aggregation of 2 μMAβ42 in the absence and presence of either 1% DMSO (black) or 0.2 (red), 0.5 (cyan), 1 (blue), 2 (yellow), 3 (green), or 5 (magenta) M eq of

UVI3003, a representative molecule of set A. The solid lines show predictions for the resulting reaction profiles when primary pathways (A,knk+), secondary pathways (B,k2k+), or both pathways (C) are inhibited by UVI3003. The abbreviation kn is the rate of primary nucleation, k+ is the rate of elongation, and k2 is the rate of secondary nucleation. Only predictions when both pathways are inhibited fit the experimental data well. The dependence of the apparent reaction rate app constants (k ) of primary pathways (D,knk+), and secondary pathways (E,k2k+), as derived from Fig. S4, is shown with increasing concentrations of small- molecule inhibitors. In each case, k represents either knk+ (primary pathways) or k2k+ (secondary pathways). (F–H) Kinetic profiles of the aggregation of a 2 μM Aβ42 solution in the absence and presence of either 1% DMSO (black) or 0.2 (red), 0.5 (cyan), 1 (blue), or 2 (yellow) M eq of CD1530, a representative molecule of

set B. The solid lines show predictions for the resulting reaction profiles when primary pathways (F,knk+), secondary pathways (G,k2k+), or both pathways (H)are inhibited by CD1530. Only predictions when both pathways are inhibited fit the experimental data well. Dependences of the apparent reaction rate constants of

primary pathways (I,knk+) and secondary pathways (J,k2k+), as derived from Fig. S5, is shown with increasing concentrations of small-molecule inhibitors.

E204 | www.pnas.org/cgi/doi/10.1073/pnas.1615613114 Habchi et al. Downloaded by guest on September 24, 2021 PNAS PLUS

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Fig. 4. Characterization of the effects of set A and set B molecules on the secondary pathways of Aβ42 aggregation. (A) Kinetic aggregation profiles of a 2 μMAβ42 solution in the presence of 10% of preformed seeds in the absence (gray) or presence of 5 M eq of bexarotene (blue), TTNPB (cyan), and UVI3003 (orchid). Under these conditions, elongation of the fibrils is the dominant mechanism. (B) Normalized growth rate constants derived from the fitted curves in A in the presence of 10% of preformed seed fibrils; these results show that set A molecules do not detectably affect the elongation rates of Aβ42 aggregation. (C) Kinetic profiles of the aggregation of a 2 μMAβ42 solution in the presence of 10% of preformed seeds in the absence (gray) or presence of 5 M eq of MM11253 (green), adapalene (blue), and BMS493 (moss green); under these conditions, elongation of the fibrils is the dominant mechanism. (D) Normalized NEUROSCIENCE growth rate constants derived from the fitted curves in C in the presence of 10% of preformed seed fibrils; these results show that set B molecules affect the elongation rates of Aβ42 aggregation at 5 M eq. (E) Kinetic profiles of the aggregation of a 2 μMAβ42 solution in the presence of 10% of preformed seeds in the absence (gray) or presence of 0.5 M eq of MM11253 (green), adapalene (blue), and BMS493 (moss green); under these conditions, elongation of the fibrils is the dominant mechanism. (F) Normalized growth rate constants derived from the fitted curves in E in the presence of 10% of preformed seed fibrils; these results show that set B molecules at 0.5 M eq do not affect the elongation rates of Aβ42 aggregation. Effect of 0.5 and 5 M eq of set A (G; bexarotene, TTNPB,

and UVI3003) and set B (H; MM11253, adapalene, and BMS493) on the rates of the surface-catalyzed secondary nucleation (k2). The rate constants were obtained from the aggregation kinetics of a 2 μMAβ42 solution in the presence of 1%, 2%, and 5% of preformed seeds (Figs. S8 and S9), where primary nucleation is negligible and surface-catalyzed secondary nucleation contributes ∼35%, 60%, and 80%, respectively, of the total quantity of fibrils formed, according to the simulations shown in Fig. S7. The quantitative parameters were obtained from the fitted curves in Figs. S7 and S8. The observed effects could only be due to decreasing the rate constants of surface-catalyzed secondary nucleation because elongation is not affected by the compounds under these conditions.

This interpretation was confirmed quantitatively by deriving the acterize the effects of the small molecules on the individual growth rate constants from the kinetic curves in the absence and microscopic steps; these effects might not otherwise be detected presence of each of the set A molecules (Fig. 4B). The molecules in directly from the aggregation kinetics in the absence of preformed set B decreased the growth rate constants by about 25% at a seeds (23, 25). Data at 1% and 5% of seeds showed a concen- concentration fivefold greater than the peptide (Fig. 4 C and D), tration-dependent inhibition of secondary pathways (i.e., a re- β thus explaining the increased delay in A 42 aggregation in the duction of k k+)ofAβ42 aggregation in the presence of both sets β 2 presence of set B molecules with respect to A 42 aggregation in A and B of compounds (Fig. 4 G and H and Figs. S8 and S9). In set A molecules. Additionally, at substoichiometric concentrations the case of set A molecules, the decrease at 0.5 and 5 M eq could (e.g., a concentration ratio of 0.5 compared with the peptide), no be attributed solely to a decrease in the rate constant of the effects were observed with any of the set B molecules on the surface-catalyzed secondary nucleation (i.e., k ) because no ef- elongation of Aβ42 fibrils, thus indicating that the inhibition of the 2 fect could be observed on the elongation of the fibrils (i.e., k+)at elongation step requires higher concentrations of small molecules molar equivalents as high as 5 (Fig. 4 A and B). The rate con- than the nucleation steps (Fig. 4 E and F). stants could be derived quantitatively from the kinetic curves and To obtain a more complete comparative assessment on the were found to be decreased by about 50% and 75% in the effects of set A and set B molecules on the secondary pathways of Aβ42 aggregation, we measured the aggregation kinetics of a presence of 0.5 and 5 M eq of set A molecules, respectively. Although the elongation of fibrils is essentially unaffected by 2 μMAβ42 sample in the presence of 1%, 2%, and 5% of fibril the set A molecules, these compounds have large effects on the seeds (Figs. S7 and S8). Simulations based on the experimental β kinetic curves show that primary nucleation is completely nucleation steps in A 42 aggregation. The effects on secondary bypassed when even the smallest ratios (1%) of preformed seeds nucleation were very pronounced in the presence of low concen- are introduced in the solution. By contrast, surface-catalyzed trations of the molecules, further supporting the key role of this β secondary nucleation and elongation contribute in different ways process in promoting the catalytic cycle in A 42 aggregation (24). to the overall kinetics, with the contribution of elongation be- By contrast, for set B molecules, the effects on the secondary nu- coming more significant with increasing seed concentrations cleation rate constant could be quantified only under the conditions (Fig. S7). Hence, following the aggregation kinetics of Aβ42 where elongation was not affected (i.e., in the presence of 0.5 M eq using different seed concentrations allows decoupling of the of the small molecules). Set B molecules were found to be signifi- secondary pathways into the surface-catalyzed secondary nucle- cantly more effective in inhibiting secondary nucleation in Aβ42 ation and elongation steps. Such decoupling is crucial to char- aggregation than set A molecules, with the decrease being as high as

Habchi et al. PNAS | Published online December 23, 2016 | E205 Downloaded by guest on September 24, 2021 A Primary nucleation Secondary nucleation Elongation PossibleTarget species B Secondary k Monomer n oligomers MM11253 k2 I Primary RAR and RXR I k Inhibitor UVI3003 modulators + oligomers I I Fibrils BMS493

LE135 BMS194753 bexarotene

adapalene Aromaticity bexarotene TTNPB Ch55 CD1530 tamibarotene I TTNPB I I BMS195614 BMS195614 UVI3003 Lipophilicity Set A molecules Set A SR11237 Ch55 SR11237 tamibarotene

MM11253 I I BMS493 I I adapalene I BMS753 LE135 I Set B molecules CD1530

Preferential sequential binding van der Waals Volume

Fig. 5. Characteristic chemical features of the small molecules found in this study to inhibit Aβ42 aggregation. (A) Summary of the effects of the tested molecules on the microscopic steps of Aβ42 aggregation and of the possible target species. (B) Correlation between lipophilicity, van der Waals volume, and aromaticity of the small molecules and their contribution to the relative potency of the molecules. The box in the figure includes the molecules from set B.

75% at 0.5 M eq, thus further confirming the higher potency of set from interactions with Aβ42 monomers. Indeed, because the mo- B molecules with respect to the potency of set A molecules. nomeric species of Aβ42 are involved in all microscopic events that underlie its aggregation, as seen in Fig. 5A (i.e., primary nucleation, Characteristic Chemical Features of Small Molecules Are Required to surface-catalyzed secondary nucleation, elongation), one possible Inhibit Specific Microscopic Steps in Aβ42 Aggregation. The obser- scenario, in which the small molecule inhibits all three steps, is that vation that set A and set B molecules affect different microscopic it binds to the common species (i.e., the monomer in all cases). We steps in the aggregation of Aβ42 could result from the interac- examined the likelihood of this scenario in a quantitative manner tions of the small molecules with different Aβ42 species formed by attempting a description of the kinetic data where the small along the aggregation pathway (Fig. 5A), because inhibiting both molecules sequester monomeric species of Aβ42. In this case, the primary and secondary nucleation could result from binding of kinetic curves in the presence of increasing concentrations of the the small molecules to primary and secondary nuclei and/or, in small molecules would correspond to a decreased concentration the latter case, fibril surfaces. On the other hand, considering the of Aβ42 available to aggregate. We found, however, that this as- qualitative correlation between the inhibited processes and the sumption is incorrect because the kinetic data could not be accu- target species, one might expect that the inhibition of Aβ42 ag- rately described (Fig. S10 A–F). Furthermore, NMR spectroscopy gregation by set B molecules may, in principle, originate mainly measurements showed no significant perturbations of the chemical

Measurements of the effect of selected molecules on Aβ42 aggregation under physiological conditions

Cerebrospinal fluid C. elegans fitness

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Fig. 6. Characterization of the effects of small molecules in CSF solutions and in a C. elegans model of Aβ42-mediated cytotoxicity. (A) Dose-dependent effects of selected molecules from set A and set B on the half-times of the aggregation reaction of Aβ42 in 66% CSF solutions. Measurements of the effects of a10μM solution of selected molecules from set A and set B in 0.6% DMSO on the frequency of body bends (B) and the paralysis rate (C) at day 5 of adulthood

of the Aβ42-worm model. Experimental data are averaged over three separate experiments (ntot ≅ 800 worms). The efficacy of these compounds was determined based on their ability to increase the fitness of the worms with respect to untreated Aβ42 worms. Statistics were determined using one-way ANOVA against 0.6% DMSO motility: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Error bars represent the SEM. BPM, bends per minute.

E206 | www.pnas.org/cgi/doi/10.1073/pnas.1615613114 Habchi et al. Downloaded by guest on September 24, 2021 shifts and the resonance intensities in the heteronuclear single cycle (i.e., L4), where no aggregation of Aβ42 has occurred yet. PNAS PLUS quantum coherence spectra of a 25 μMsampleof15N-labeled At day 5 of worm adulthood, where the fitness of the Aβ-worms monomeric Aβ42 before and after the addition of each of the set B was significantly decreased compared with the control-worms molecules and of UVI3003, the most effective molecule of set A, as (Fig. S14A), the selected small molecules restored the motility of a control (Fig. S10 G–L). These observations indicate that set B the worms substantially, except for the negative control BMS753 molecules are likely to bind both primary and secondary nuclei, (Fig. 6B), in a concentration-dependent manner (Fig. S14B). and the fibril surfaces and ends, but not monomers. These results are similar to the results observed in vitro and in Unlike set A molecules, set B molecules were found to inhibit human CSF. In addition, no selected molecules showed any all three major steps in Aβ42 aggregation, with the effects on the significant effect in a wild-type worm model that does not ex- primary and secondary nucleation steps being greater than the press Aβ42, N2 (termed the control-worm model) (Fig. S14C). effects of the set A molecules. It is thus of great significance to These data suggest that the small molecules restore the motility identify the chemical features responsible for these differences, of the Aβ-worms by specifically inhibiting the aggregation of because such features can at least in principle enable the rational Aβ42. Indeed, the level of aggregates was measured in Aβ-worms design of molecules against specific microscopic steps in Aβ42 in the absence and presence of bexarotene using the fluorescence aggregation. From an analysis of the molecular properties of the intensity of the amyloid-specific dye NIAD-4, and was found to small molecules, we selected two parameters: the lipophilicity, be similar to the level of aggregates of the control-worms in the defined as the Ghose–Crippen octanol/water coefficient (30), presence of bexarotene, where no aggregates could be detected and the steric bulk, defined as the sum of the atomic van der (22). We found that all selected small molecules decreased the Waals volumes (31). We identified a linear correlation between rate of paralysis of the Aβ worms, except for MM11253. This these two parameters that largely accounts for the efficacy of all finding is in agreement with the unexpected lower increase in of the small molecules (Fig. 5B). The greater the lipophilicity and the the motility of the Aβ-worms in the presence of MM11253, steric bulk, the higher is the potency of the small molecule, with very which was found to be extremely potent in vitro. Because no low values of their sum corresponding to the complete abolition MM11253-related toxicity could be observed, as judged from the of the effects of a molecule, as in the case of BMS753. Strikingly, absence of any effect on the fitness of control worms (Fig. S14C), UVI3003, the most potent molecule in set A, was found to possess this finding is likely to be due to a lower half-life of this molecule similar lipophilicity and steric bulk values to those values in set B in worms compared with adapalene, bexarotene, and UVI3003 molecules, and, accordingly, a similar potency is predicted from (Fig. 6C). In any case, all molecules that have shown an effect on

the correlation in Fig. 5B. Although no effect was observed on the the aggregation of Aβ42 in vitro are also able to restore the NEUROSCIENCE elongation step, UVI3003 inhibits both the primary and secondary fitness of the Aβ-worms, thus further supporting the power of nucleation steps to a similar degree to the molecules in set B. the kinetic assay in drug discovery. The effects of the small These results suggest that the lipophilicity and steric bulk describe molecules are also readily detectable under stress conditions, well the effects of small molecules on the primary and secondary because bexarotene (set A) and adapalene (set B) restore the nucleation steps, but not on the elongation step. To decipher the fitness, in terms of motility (Fig. S15A) and paralysis (Fig. chemical features of the small molecules responsible for inhibiting S15B), of Aβ-worms that were exposed to temperature-induced the elongation step, we considered an additional parameter: the stress (Fig. S15). value of the relative aromaticity of the small molecules, defined as the ratio of the number of aromatic atoms to the total number of Conclusions atoms. We thus found that a high aromaticity value confers the We have described a drug discovery approach based on quanti- ability to inhibit the elongation of Aβ42 fibrils (Figs. 4 C and D tative chemical kinetics to identify small molecules that target and 5B). Indeed, all of the set B molecules have aromaticity values specific microscopic steps in Aβ42 aggregation. The results that greater than the aromaticity values of the set A molecules. we have obtained suggest that this approach is highly effective for drug discovery against protein misfolding diseases because, RAR and RXR Ligands Inhibit Aβ42 Aggregation in CSF Solutions and unlike other available approaches not based on chemical kinet- Rescue Aβ42-Mediated Dysfunction in a C. elegans Model. We next ics, it provides quantitative descriptions of the inhibitory process, explored if the small molecules retard Aβ aggregation under more thus making it possible to control and modulate the onset of the physiologically relevant conditions. We therefore monitored these aggregation in vitro. In addition, the molecules that we have effects on the aggregation kinetics of Aβ42 in human cerebro- described, particularly those in set B, are expected to have a spinal fluid (CSF). CSF caused a concentration-dependent re- greater effect not only on the onset of aggregation but also on tardation of Aβ42 aggregation, suggesting that Aβ42 aggregation is the proliferation of the Aβ42 oligomers produced through surface- slower in this fluid, in line with previous results (32) (Fig. S11A). catalyzed secondary nucleation. Therefore, targeting such a pro- We then investigated the effects of selected small molecules from cess, which is largely responsible for the production of toxic Aβ42 set A (bexarotene and UVI3003) and set B (MM11253 and ada- species (23, 24), should provide an effective means for the devel- palene), and from BMS753 as a negative control, under conditions opment of treatments against the progression of AD. Given the where the effect of CSF is close to maximal (i.e., 66%) (Fig. connection between the aggregation of Aβ42 and AD, and the fact S11B). We found that under these conditions, all selected small that the typical age for the onset of sporadic AD is around 65 years, molecules, except for BMS753 (Fig. S12), significantly delayed the even a small delay in Aβ aggregation may postpone the onset by aggregation kinetics in a concentration-dependent manner similar long enough to reduce the risk of developing AD significantly. to what has been observed in buffer (Fig. 6A and Fig. S13). Thus, inhibiting the nucleation steps in Aβ42 aggregation should We further evaluated the effects of the same small molecules result in a delay and reduction in the formation of toxic Aβ42 in a C. elegans model of Aβ42-mediated dysfunction, denoted oligomers (22, 23), which are considered central to the pathology of GMC101 (termed the Aβ-worm model) (22). In this model, AD; therefore it is likely to be a promising route to preventing AD. Aβ42 is expressed in body wall muscle cells, where it forms ag- We anticipate that the combination of QBSDD and kinetics-based gregates and results in progressive paralysis (22). We assessed drug discovery will allow the screening of databases for the identi- the effects of the small molecules on the fitness of the worms in fication of pools of potent small molecules against Aβ42 aggrega- terms of the frequency of body bends (i.e., motility) and the rate tion. This approach will also enable the rational design of candidate of their paralysis. Because all molecules from both sets A and B molecules bearing the chemical features that are crucial for inhib- have significant effects on the nucleation steps of Aβ42 aggre- iting specific microscopic steps of the protein aggregation reaction, gation, we added them at the larval stages of the C. elegans life and that possess good drug pharmacokinetic characteristics.

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E208 | www.pnas.org/cgi/doi/10.1073/pnas.1615613114 Habchi et al. Downloaded by guest on September 24, 2021