Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer

Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer Jerson L. Silva,*,† Elio A. Cino,‡ Iaci N. Soares,† Vitor F. Ferreira,§ and Guilherme A. P. de Oliveira*,†,∥ †

Instituto de Bioquı ́mica Médica Leopoldo de Meis, Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil ‡ Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Brazil § Departamento de Tecnologia Farmacêutica, Faculdade de Farmácia, Universidade Federal Fluminense, 24220-900 Rio de Janeiro, Brazil ∥ Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908-0733, United States CONSPECTUS: Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer’s, Parkinson’s, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53−MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73. Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor−acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and combined efforts, the outlook is positive for the development of strategies for regulating p53 amyloid transformation. its cancer-associated mutants.1,2 Although the number of independent findings supporting the aggregation of mutant p53 in cancer is rapidly expanding,1−5 it is still not fully accepted that blocking the amyloidogenic route of p53 might

I. INTRODUCTION The impairment of p53 tumor suppressor functions originated by mutations frequently present in cancers has motivated an array of potential therapeutic approaches. In the last 20 years, our group and others have provided compelling evidence of the amyloidogenic prion-like behavior (i.e., cell-to-cell transmissibility and seeded aggregation) of wt p53 and several of © XXXX American Chemical Society

Received: September 26, 2017

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DOI: 10.1021/acs.accounts.7b00473 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Figure 1. Multiple facets of p53. (a) p53 in normal cells is tightly controlled and degraded through the proteasome. (b) Upon genotoxic stress, p53 is activated and assumes tumor suppressor functions. (c) Several mechanisms including the LoF, GoF and DN activity of p53 assist with tumor progression. (d) p53 aggregation and sequestration of other proteins, leading to GoF. (e) As a prionoid, p53 spreads to other cells and the sequestration of normal p53 maintains the tumor. Inactive p53 is colored gray.

Figure 2. P53 mutations in cancer and aggregation segments. Tumor site distribution of all p53 somatic mutations (according to the last updated version of the TP53 mutant database, http://p53.iarc.fr, on april 2016). Codon distribution of somatic mutations highlighting the hotspot variants within the DNA-binding domain (adapted from http://p53.iarc.fr). Aggregation-prone segments are red labeled. TAD: Transactivation domain, DBD: DNA-binding domain, PPR: polyproline region, OD: Oligomerization domain.

be a promising target for anticancer therapy. p53 aggregation in cancer is an ongoing topic of fundamental research but is often overlooked by cancer and amyloid research communities. The recent concept of p53 as a prionoid,6,7 and the fact that several tumor types have not been explored yet for the p53 amyloid signature, may contribute to this skepticism.

In this Account, we (i) review the loss-of-function (LoF), dominant negative activity (DN), and gain-of-function (GoF) effects of prion-like mutant p53 aggregation in cancer; (ii) discuss current challenges in preventing p53 aggregation, including the use of small molecules with anticancer potential; (iii) describe our methodology for trapping aggregation precursor states in solution, and (iv) discuss how the results B


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Figure 3. Aggregation reaction of p53. Folded p53 undergoes conformational changes and populates competent states that may form different types of aggregates, including oligomers, amorphous aggregates, and amyloid fibrils. The participation of liquid−liquid phase transitions (LLPT) upon binding to RNA and p53 fibril strains are highlighted as possible mechanisms in the p53 aggregation scheme. Cofactors such as glycosaminoglycans (GAGs), e.g., chondroitin sulfate (CsA) may also play a role in p53 fibril formation. Targeting preamyloidogenic competent states of p53 may represent a potential strategy to block p53 aggregation in cancer.

from trapping experiments, together with those from MD simulations, can be used to understand the molecular basis of p53 aggregation, and applied to interfere with its amyloid conversion.

P53 aggregates form heterogeneous structures of amorphous assemblies, oligomers, and amyloid-like fibrils (Figure 3). Amorphous aggregates are formed by a combination of mutant and wt p53 and may contain other proteins, including the p53 family members p63 and p73.15,16 Sequestration with other proteins leading to GoF could occur by allowing p53 to bind different responsive elements and regulate new target genes, or avoidance of DNA binding and triggering of new signal cascades by the aggregates (Figure 1d). For instance, several proteins with the ability to bind p53 aggregates were identified in ovarian cancer stem cells, and participate in exocytosis, cytokinesis, metabolism, ribosome biogenesis, and RNA processing.17 As discussed below, our results suggest that aggregation of mutant p53 and its seeding properties are possible GoF mechanisms participating in cancer (Figure 1d).1,15,17

II. PRION-LIKE p53 AGGREGATION IN CANCER Tumor Suppressor or Oncogene?

The p53 transcriptional factor is tightly controlled by the E3 ubiquitin protein ligase MDM2 (Figure 1a).8 Upon exposure to ionizing radiation, p53 degradation becomes disfavored, promoting cell-cycle arrest, senescence, and apoptosis, preventing tumor occurrence (Figure 1b). Conversely, p53 functions are modified when p53 is overexpressed or inactivated by mutations. More than 31 000 p53 somatic mutations in human cancer have been identified at the time of this publication (p53.iarc.fr). Over 95% of these mutations occur in the DNA-binding domain (DBD) (Figure 2) and affect either the ability to bind DNA (contact mutants) or the structural stability (structural mutants).9 Monoallelic mutations in p53 have shown a dominant-negative activity of the mutant protein to inactivate the wt and further explain its loss-offunction (LoF) (Figure 1c).10 Mutations can also transform p53 into a malignant oncogene with gain-of-function (GoF) phenotypes including invasion, migration, angiogenesis, proliferation, tissue remodeling, and chemoresistance, with different mechanisms proposed to explain these new functions.9,11 Different p53 mutants in diverse tumor types activate different p53 target genes, but some may give rise to the same GoF phenotype.12,13 For example, M237I, in glioblastoma, and R282W, in lung cancer, trigger chemoresistance of cells through the activation of distinct p53 downstream genes.12,13 Additionally, binding of p53 GoF mutants to chromatin regulatory genes, such as methyltransferases (MLL1 and MLL2) and acetyltransferase (MOZ), lead to the genomic increase of histone methylation and acetylation, two important modifications participating in gene transcription regulation.14

Cancer as an Amyloidogenic Disease

Deposition of amyloids is the hallmark of neurodegenerative disorders.18 The identification of sequences prone to amyloid aggregation and assessment of their roles in normal function, as well as disease establishment, progression and spreading are among the prominent topics discussed in recent literature.19 Amyloids were shown to have a role in physiological activities such as skin pigmentation,20 storage of protein hormones in secretory granules21 and the fixation of long-term memory.22 In the early 1980s, Stanley Prusiner’s group provided evidence for a class of diseases called TSEs. They have shown that the infectious agent is formed solely by a misfolded protein, which they termed a prion.23 Specific mutations in this protein lead to unique neuropathological disorders providing the phenotypic variability of prion diseases.24 The fundamental mechanism of prion infectivity is template conversion, host transmissibility, and seeded aggregation, in which a misfolded protein is generated, and passed to other cells and organisms. This prion concept was expanded to a series of other amyloidogenic proteins involved in senile disorders.25 A general and destructive feature of these prionoids is the self-assembly of C


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Figure 4. Potential molecules to target p53 aggregation, stability, and interaction with MDM2. (a−e) Different classes of small-molecule compounds at various stages of therapeutic development. Some have already been explored for their ability to stop p53 aggregation, and others are still awaiting further investigation. Red arrows indicate possible centers of nucleophilic attack on Michael’s acceptor.

through classical seeding but instead, coaggregation.30 Our findings suggest coaggregation or seeding will depend on the stoichiometry of mutant and wt proteins.1 The etiologic agent that converts prion proteins to their misfolded and infectious forms is yet unexplored, but cofactors may play a role during conversion.31−33 In the case of p53, the in vitro ability of seeds to accelerate aggregation, was recently confirmed by Maji’s group for the full-length protein using cellbased assays.2 In agreement with the cofactor hypothesis to explain the etiology of “prion” conversion,34,35 p53 was shown to bind RNA, in addition to DNA. Recently, we described for the first time the modulation of p53 aggregation and seeding by RNA molecules.36 Our observations might suggest that p53 can associate with membrane-less organelles formed by RNA molecules and disordered, low-complexity regions of RNAbinding proteins.37 The mechanism for assembling these organelles is one of liquid−liquid phase transitions (LLPT) which are commonly related to neurodegenerative disorders.38 Stress granules of several disease-related RNA-binding proteins, such as TDP-43 and FUS, require LLPT that evolve to amyloid fibrils.38 Nuclear inclusion bodies formed by mutant and wt p53 have been detected in tumor biopsies and related to

host-derived proteins that fail to incite the host immune system, allowing extended periods of incubation, fibril growth, and dissemination before symptoms appear. We have shown the cytotoxicity of p53 aggregates upon exposure to culture cells1 but the signaling cascade triggered in these cells remains to be uncovered. The in vitro formation of p53 fibrillar structures26 was shown for the first time in 2003 by our group. Subsequently, we confirmed its amyloidogenic properties in cancer cells and breast-cancer biopsies,27 and proposed the prion-like behavior of p53.1 The assumption of a prion-like phenotype for p53 was first proposed in 2012 based on the ability of a mutant p53 to seed the wt p53 in vitro. Following our discoveries, several groups have identified aggregated forms of p53 in different tumor types.2−4,15,17,28 The oligomeric and fibrillar species of p53 are commonly identified in cell-based assays using antibodies for generic amyloidogenic epitopes.29 The isolation of tissue-derived fibrils of p53 from human tumors and the prion-like cell-to-cell transmission of p53 provided further support for the view that cancer has fundamental features of prion diseases (Figure 1e).2 However, kinetic studies by Wang and Fersht showed that p53 aggregation does not evolve D


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Accounts of Chemical Research microenvironment stress.4 However, LLPT composed of p53 and RNA, along with its implications for cancer, has yet to be shown. Another important concept is that of amyloidogenic strains, and their role in distinct disorders.39 Similar to the heterogeneous nature of intermediate species (e.g., oligomers), mature amyloid filaments also present considerable structural diversity. Many studies suggest that the amyloid polymorphism of aggregates may dictate disparities in clinical characteristics of neurodegenerative diseases.39,40 The understanding of how structural polymorphs or strains are linked to specific disease subtypes will be a prerequisite for future directions on the therapeutics of neurological disorders. The likely existence of strains of amyloid aggregates of p53 remains to be investigated in detail.

Reactivation of p53 Function by Small Molecules

to determine its highest feasible dose for intravenous administration in patients with refractory hematologic malignancies or prostate carcinoma. Our group has evidence that PRIMA-1 may act by inhibiting mutant p53 aggregation.50 Sulfonylpyrimidines restore tumor suppressor function of mutant p53. Ligands containing an α,β-unsaturated double bond, bind covalently to generic Cys sites in the core domain and increase the thermostability of wt and mutant p53.51 CP31398 stabilizes p53 activity in cancer cell lines (Figure 4b). It inhibited the growth of small human tumor xenografts in vivo and tobacco-induced lung cancer52 and became a potential chemo preventive agent for bladder cancer. Such compounds have low cytotoxicity to healthy cells and are not general alkylating agents.53 A novel small molecule named MPK-09 (Figure 4b) proved to be very selective and highly potent in restoring functions of R175H, R249S, R273H, R273C, and E285K mutants.54 The compound PK7088 was shown to bind the p53 Y220C mutant in cancer cells, inducing growth inhibition, cell-cycle arrest and apoptosis; it works synergistically with Nutlin-3 to increase the amount of folded p53.55 The design of compounds based on the Nutlin structure aims to reactivate mutant p53, either through interaction with p53 pockets or alkylation of thiols.49,55 Several maleimide derivatives can reactivate DNA binding of mutant p53, preserving its active conformation.56 The compound PK5174 was able to prevent Y220C aggregation47 while STIMA-1 recapitulated p53 biological activity by alkylating thiol groups (Figure 4c).57 In 2004, Nutlins were reported as potent and specific inhibitors for p53-MDM2 interaction.58 Nutlin-2 and Nutlin-3a have the highest affinity for recombinant p53-MDM2, with IC50 of 140 and 90 nM, respectively. Crystal structures of Nutlin-2 with p53-MDM2 revealed a three-finger pharmacophore model of interaction. A Nutlin-3a derivative compound such as RG7112 (Figure 4d) stabilizes p53 and has a 3-fold higher effect than Nutlin-3a in inhibiting cell growth.59 It entered phase I clinical trials combined with cytarabine in participants with acute myelogenous leukemia, neoplasms, and hematologic neoplasms. Some compounds seem to have multiple mechanisms of actions to reactivate p53 function, such as RITA.60 It inhibits the MDM2-p53 interaction by targeting R175H, R248W, R273H, and R280K, restoring transcriptional activity and inducing apoptosis (Figure 4e). Stictic acid was selected as a potential p53 reactivator by targeting Cys124 (Figure 4e).49 In fact, it exhibits p53 R175H mutant reactivation in human osteosarcoma cells and dose-dependent expression of p21. The use of some natural polyphenols that have antiamyloid effects is another strategy. Resveratrol clears Aβ plaques in mouse models of AD.61 We have demonstrated that resveratrol inhibits cancer development through induction of p53-dependent cell death.62,63 More recently, we found that resveratrol has a significant antiamyloid effect on mutant p53.

Some alkylating agents cause permanent changes in the p53 structure by reacting with Cys124 and Cys141 but not compromising its DNA-binding activity. Compounds interacting with the L1/S3 pocket, a region localized around Cys124, Cys135, and Cys141, cause small structural changes and reactivate mutant p53.49 PRIMA-1 and PRIMA-1Met are the most successful compounds causing p53 reactivation; they react covalently with thiol groups of mutant p53, leading to apoptosis (Figure 4a).43,44 PRIMA-1Met is currently in clinical phase I/II

IV. TRAPPING AGGREGATION PRECURSOR STATES OF p53 We have shown that aggregation-competent p53 conformers carry an increased β content,64 and the use of Φ-values to evaluate the early transition states of p53 aggregation has provided insights into p53 amyloidogenic conversion.65 Indeed, p53 aggregation may proceed through multiple sites in its DNA-binding domain (DBD).66,67 As such, interfering with

III. TARGETING p53 AGGREGATION IN CANCER THERAPY P53 Therapies

Approaches to retrieve p53 function in cancer include the use of small-molecule or peptide stabilizers of misfolded p53, zinc administration, gene therapy, metallochaperones, alkylating and DNA intercalators, blockage of p53-MDM2/X interaction, and other p53 regulators.19,41,42 A peptide that interacts with the p53 segment that exhibits the highest propensity to aggregate has been tested in ovarian carcinomas with p53 mutations and was able to rescue p53 function.42 Some anticancer compounds such as PRIMA-1Met bind the DNA-binding domain of p53,43,44 while others, such as Nutlins, interfere with the p53-MDM2 interaction (Figure 4). In hepatocellular carcinoma, the destabilized Y220C mutant is a good target due to a surface cavity introduction.45 This mutant has been extensively screened for small-molecule stabilizers using virtual- and fragment-library screening and structure-guided design.46 Y220C aggregation is also inhibited with designed ligands.47 Although small-molecule inhibitors are being explored as a potential therapy against p53 aggregates, the extensive repertoire of cancer-associated p53 mutations complicate this approach. After achieving mature aggregates and fibrils, the search for druggable surfaces might represent an ineffective strategy because potential small-molecule candidates would tend to stabilize instead of dissociating the assemblages. A potential solution might be to block p53 aggregation by targeting the intermediate species of the aggregation reaction, such as the oligomers, or the conformers populated at the nucleating steps, in which conformational changes in folded p53 take place (Figure 3). Understanding of p53 dynamics and how transient48 and aggregation-competent states are formed represent a crucial strategy to block p53 aggregation. Trapping on-pathway GoF oligomers from the p53 aggregation reaction is a reasonable idea to avoid the building of complex assemblies observed in cancer cells.

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Figure 5. Experimental and in silico evidence of p53 preamyloidogenic states. (a,b) In vitro assays of p53 and p63 DBD aggregation and stability. (a) ThT binding assay and (b) HHP assay showing the higher tendency of p53 aggregation. (c) Diagram of phasor plots combining subdenaturing concentrations of guanidine (GnD) and HHP reveals different p53 states (unpublished data). (d−f) Insights into structurally vulnerable sites in the p53 DBD using MD. (d) BHBs in p53 and p63 DBDs. (e) Comparison of p53 and p63 BHB protection factors (negative values signify lower protection in p53 vs p63), and (f) p53 DBD structure showing the three least protected BHBs compared to p63. Adapted with permission from ref 66. Copyright 2016 Nature. DBD: DNA-binding domain, BHB: backbone hydrogen bonds, HHP: high-hydrostatic pressure, and ThT: thioflavin T.

only one site may not represent an effective strategy to block p53 aggregation. We have employed a combination of physical, chemical, and computational strategies to acquire information on the initial steps of protein aggregation (Figure 5). Our studies have shown that high hydrostatic pressure (HHP) and chemical denaturants may help to clarify features of molten-globule (MG) states that are prone to aggregation.68,69 In contrast to p63, p53 DBD rapidly forms amyloidogenic ThT-binding aggregates and is susceptible to structural perturbation by HHP (Figure 5a,b). Despite having similar sequences (∼60% identity) and structures, the results suggest the presence of individual p53 preamyloidogenic states. Deviations from linearity are detected in phasor plots of single Trp fluorescence signals of p53, supporting the presence of hidden excited state conformers with molten-globule properties specifically to p53 (Figure 5c) (unpublished data). Distinct preamyloidogenic

molten-globule species were detected under varying concentrations of denaturants and high hydrostatic pressure along the p53 aggregation pathway (Figure 5c). Additionally, we found that p53 molten-globule states are identified in cell-based assays in combination with lysosomes.70 The high hydrostatic pressure mechanism of perturbation through the pushing of water into proteins is unlike that of chemical denaturants, providing clear insights into protein stability.66,69 Protection of backbone hydrogen bonds (BHBs) by nonpolar amino acid side chain carbon atoms has been shown to be a crucial factor for keeping protein cores dry and, consequently, protein stability.71 Due to the experimental challenges in describing atom-level BHB dynamics and protection, molecular dynamics (MD) simulations have been applied to identify potentially deficient sites.66,72 For instance, solvent-exposed BHBs in p53 Y220C described by MD can be targeted by small molecules to rescue the mutant.72 These and F


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TWAS Prize in Biology, and the Science Prize from Fundaçaõ Conrado Wessel. Silva is member of the Brazilian Academy of Sciences, World Academy of Sciences (TWAS) and the National Academy of Medicine. His main research interest is in the study of the basic factors of protein misfolding and aggregation in neurodegenerative diseases and cancer. Among his most important discoveries are the amyloid aggregation of p53 and the recognition that prions and prion-like proteins have other accomplices, such as nucleic acids.

other recent MD studies uncover new roles of dynamics and hydration on p53 structure in the context of drug development.48,73 Our results show considerable differences in BHB profiles among p53 family members, with p63 and p73 DBDs exhibiting higher protection compared to p53.66 Although p53 and p63 show highly similar patterns of BHB presence (Figure 5d), they have notable differences in protection (Figure 5e). Pinpointing the donor−acceptor pairs in p53 that exhibit lower protection than p63 (Figure 5f) should help to identify sites of structural vulnerability, and provide new insights into rational designs that can be rapidly screened using our experimental methodology.

Elio A. Cino obtained his B.Sc. and Ph.D. in Biochemistry at the University of Western Ontario. He completed postdoctoral fellowships at the University of Waterloo and the Federal University of Rio de Janeiro. Currently, Elio is an Assistant Professor in the department of Biochemistry and Immunology at the Federal University of Minas Gerais where he combines experimental and computational methods to study intrinsically disordered proteins, protein stability, and protein−protein/ligand interactions.

V. CONCLUDING REMARKS Despite years of comprehensive research on numerous aspects of p53, we are presently discovering new facets of aggregated p53 and its prion-like features in cancer. The cellular responses of different aggregated forms of mutant p53, the interplay with the multitude of p53 targets and signaling to other cells and, their consequences for cancer development and progression represent a burgeoning field and several new possibilities for therapy. Independent and collaborative research on the prionlike features of p53 will overcome some of the skepticism about targeting aggregated p53 in the pipeline of therapeutic strategies. In a recent comprehensive review on p53,74 Kastenhuber and Lowe state that “TP53 mutant cancer will lead to the deaths of more than 500 million people alive today” if new therapeutic approaches are not developed.74 While targeting of mutant p53 aggregation is recognized to be an important treatment strategy, further research must be conducted to better characterize the aggregates, mechanisms, and GoF effects. It is clear that unraveling the cell biology and structural aspects of p53 amyloid aggregates will be one of the most important themes of oncobiology in the near future.

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Iaci N. Soares completed her B.Sc in Biological Sciences at Federal University of Minas Gerais (UFMG). She received her Ph.D. in Molecular and Biochemical Pharmacology at UFMG and was a postdoctoral fellow at the University of Western Ontario and Federal University of Rio de Janeiro, and is currently at UFMG Neuroscience. Her major research interests include molecular studies of protein modification and their physiological impact. Under the broad fields of neurodegenerative diseases and cancer, she applies biochemical techniques to investigate protein aggregation, interaction, and modification. Vitor Francisco Ferreira obtained his B.Sc. in chemistry in 1976 and MSc in chemistry of natural products in 1980. He was a doctoral student at the University of California San Diego and received the Hart Memorial Award for best foreign student in 1982 (UCSD). In 1990, he was appointed Associate Professor at the Universidade Federal Fluminense, and became Full Professor in 2002. Vitor Ferreira is a member of the Brazilian Academy of Science and was awarded in 2008 the Brazilian Order of Scientific Merit. His research interests include synthesis using readily available carbohydrate, synthetic methods using diazocompounds, and synthesis of bioactive compounds for neglected diseases.

AUTHOR INFORMATION

Corresponding Authors

*Mailing address: Programa de Biologia Estrutural, Instituto de Bioquı ́mica Médica Leopoldo de Meis, Instituto Nacional de Biologia Estrutural e Bioimagem, Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, RJ, Brazil. E-mail: [email protected]. *E-mail: [email protected].

Guilherme A. P. de Oliveira has a B.Sc. in Biomedicine (Honors) from the Federal University of Rio de Janeiro (UFRJ) and a M.S. and Ph.D. in Biological Chemistry and Biophysics from the Medical Biochemistry Institute (IBqM), UFRJ (Supervisor: Jerson L. Silva). Currently, he is an Associate Professor at the IBqM, UFRJ and a postdoc at the University of Virginia under a Pew Charitable Trusts fellowship (Supervisor: Edward H. Egelman). His research is devoted to basic approaches including fluorescence spectroscopy, high-pressure nuclear magnetic resonance and, cryo-EM of helical filaments and single particle analysis of amyloids in cancer and neurodegenerative disorders.

ORCID

Jerson L. Silva: 0000-0001-9523-9441 Guilherme A. P. de Oliveira: 0000-0002-0063-5888 Author Contributions

Written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

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The authors declare no competing financial interest. Biographies

ACKNOWLEDGMENTS

We thank Dr. Martha M. Sorenson for critical reading of the manuscript. Our laboratories were supported by grants from Conselho Nacional de Desenvolvimento Cientı ́fico e Tecnológico (CNPq awards and the INCT Program), Fundaçaõ Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı ́vel Superior (CAPES).

Jerson L. Silva received M.D. and Ph.D. degrees from the Universidade Federal do Rio de Janeiro (UFRJ). He is Professor of the Institute of Medical Biochemistry (UFRJ), founding director of the Jiri Jonas National Center for Nuclear Magnetic Resonance (CNRMN) and coordinator of the National Institute for Structural Biology and Biomaging (INBEB). Silva has received many prizes and awards, including Fellow of the John Simon Guggenheim Foundation, G


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