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 elonga- tion (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.