Advances in Organic Synthesis: Volume 7 E-Book
32,64 €
Mehr erfahren.
- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Serie: Advances in Organic Synthesis
- Sprache: Englisch
Advances in Organic Synthesis is a book series devoted to the latest advances in synthetic approaches towards challenging structures. It presents comprehensive articles written by eminent authorities on different synthetic approaches to selected target molecules and new methods developed to achieve specific synthetic transformations. Contributions are written by eminent scientists and each volume is edited by an authority in the field. Advances in Organic Synthesis is essential for all organic chemists in the academia and industry who wish to keep abreast of rapid and important developments in the field.
This volume presents reviews on the following topics:
Small molecules that influence protein folding
Recent advances in peptide and protein synthesis
Bioorthogonal coupling for imaging and radiotherapy
Xanthenedione synthesis
Heterogeneous catalysis in organic synthesis
Sie lesen das E-Book in den Legimi-Apps auf:
Seitenzahl: 536
Veröffentlichungsjahr: 2017
Ähnliche
Advances in Organic Synthesis
(Volume 7)
Edited by
Atta-ur-Rahman, FRS
BENTHAM SCIENCE PUBLISHERS LTD.
End User License Agreement (for non-institutional, personal use)
This is an agreement between you and Bentham Science Publishers Ltd. Please read this License Agreement carefully before using the ebook/echapter/ejournal (“Work”). Your use of the Work constitutes your agreement to the terms and conditions set forth in this License Agreement. If you do not agree to these terms and conditions then you should not use the Work.
Bentham Science Publishers agrees to grant you a non-exclusive, non-transferable limited license to use the Work subject to and in accordance with the following terms and conditions. This License Agreement is for non-library, personal use only. For a library / institutional / multi user license in respect of the Work, please contact: [email protected].
Usage Rules:
All rights reserved: The Work is the subject of copyright and Bentham Science Publishers either owns the Work (and the copyright in it) or is licensed to distribute the Work. You shall not copy, reproduce, modify, remove, delete, augment, add to, publish, transmit, sell, resell, create derivative works from, or in any way exploit the Work or make the Work available for others to do any of the same, in any form or by any means, in whole or in part, in each case without the prior written permission of Bentham Science Publishers, unless stated otherwise in this License Agreement.You may download a copy of the Work on one occasion to one personal computer (including tablet, laptop, desktop, or other such devices). You may make one back-up copy of the Work to avoid losing it. The following DRM (Digital Rights Management) policy may also be applicable to the Work at Bentham Science Publishers’ election, acting in its sole discretion:25 ‘copy’ commands can be executed every 7 days in respect of the Work. The text selected for copying cannot extend to more than a single page. Each time a text ‘copy’ command is executed, irrespective of whether the text selection is made from within one page or from separate pages, it will be considered as a separate / individual ‘copy’ command.25 pages only from the Work can be printed every 7 days.3. The unauthorised use or distribution of copyrighted or other proprietary content is illegal and could subject you to liability for substantial money damages. You will be liable for any damage resulting from your misuse of the Work or any violation of this License Agreement, including any infringement by you of copyrights or proprietary rights.
Disclaimer:
Bentham Science Publishers does not guarantee that the information in the Work is error-free, or warrant that it will meet your requirements or that access to the Work will be uninterrupted or error-free. The Work is provided "as is" without warranty of any kind, either express or implied or statutory, including, without limitation, implied warranties of merchantability and fitness for a particular purpose. The entire risk as to the results and performance of the Work is assumed by you. No responsibility is assumed by Bentham Science Publishers, its staff, editors and/or authors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products instruction, advertisements or ideas contained in the Work.
Limitation of Liability:
In no event will Bentham Science Publishers, its staff, editors and/or authors, be liable for any damages, including, without limitation, special, incidental and/or consequential damages and/or damages for lost data and/or profits arising out of (whether directly or indirectly) the use or inability to use the Work. The entire liability of Bentham Science Publishers shall be limited to the amount actually paid by you for the Work.
General:
Any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims) will be governed by and construed in accordance with the laws of the U.A.E. as applied in the Emirate of Dubai. Each party agrees that the courts of the Emirate of Dubai shall have exclusive jurisdiction to settle any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims).Your rights under this License Agreement will automatically terminate without notice and without the need for a court order if at any point you breach any terms of this License Agreement. In no event will any delay or failure by Bentham Science Publishers in enforcing your compliance with this License Agreement constitute a waiver of any of its rights.You acknowledge that you have read this License Agreement, and agree to be bound by its terms and conditions. To the extent that any other terms and conditions presented on any website of Bentham Science Publishers conflict with, or are inconsistent with, the terms and conditions set out in this License Agreement, you acknowledge that the terms and conditions set out in this License Agreement shall prevail.Bentham Science Publishers Ltd. Executive Suite Y - 2 PO Box 7917, Saif Zone Sharjah, U.A.E. Email: [email protected]
PREFACE
This volume of Advances in Organic Synthesis presents some recent exciting developments in synthetic organic chemistry. It covers a range of topics including important researches on novel approaches to the construction of complex organic compounds. The book should prove to be a valuable resource for pharmaceutical scientists and postgraduate students seeking updated and critically important information in synthetic organic chemistry. The chapters are written by authorities in the field and are mainly focused on protein mis-folding using small molecules, proteins and peptides synthesis, pretargeted nuclear imaging and radiotherapy, and the role of catalysts in organic synthesis. I hope that the readers will find these reviews valuable and thought provoking so that they may trigger further research in the quest for new developments in the field.
I am grateful to Bentham Science Publishers for the timely efforts made by the editorial personnel, especially Mr. Mahmood Alam (Director Publications), Mr. Shehzad Naqvi (Senior Manager) and Dr. Faryal Sami (Assistant Manager).
List of Contributors
Small Molecules that Ameliorate Protein Mis-folding
Paolo Ruzza*Abstract
Protein misfolding is characterized by the inability of proteins to achieve or maintain their bioactive conformation. In addition to protein mutation, intracellular factors such as pH changes, metal ions, and oxidative stress contribute to protein misfolding. To modulate the level of misfolded proteins, different approaches are feasible including the use of pharmacological or chemical chaperones, the activation of degradative pathways and the manipulation of natural folding mechanism. Errors in protein folding are correlated to a broad range of diseases, from common allergies to neurodegenerative diseases, and at the moment, many examples exist of the successful control of protein unfolding that may be used in the therapy of these disorders. This chapter gives an overview on small molecules that can be used to stabilize protein, helping it to achieve near-native conformation and bring back its functions with an emphasis on pharmacological and chemical chaperones.
INTRODUCTION
The successful execution of biological function of proteins depends on their correct folding into well-defined three-dimensional structures. The folded three-dimensional structures of most proteins represent a compromise between thermodynamic stability and conformational flexibility required for their function. Consequently, proteins are often marginally stable in their physiological environment. Moreover, a fraction of proteins (about 30%, in eukaryotic cells) is classified as intrinsically unstructured. Amino acid mutations strongly increase the propensity of a protein to misfold, even if some wild type proteins have a high tendency to misfold, and intracellular factors such as pH, oxidative stress, and metal ions may play an important role in the induction of an incorrect protein
folding [1]. Protein misfolding is strongly implicated in aging and in the patho-genesis of different well-known diseases including cystic fibrosis, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS), as well as in disorders such as Gaucher’s disease, nephrogenic diabetes insipidus, and Creutzfeldt-Jakob disease [2].
Diseases related to protein misfolding occur via two different pathways correlated to either a loss of protein function (LOF) or a gain of new protein function (GOF) (Fig. 1).
Fig. (1)) Schematic illustration of protein folding and protein misfolding diseases pathways. Loss of Function (LOF) arises from the inability of a partially folded protein to achieve its functional conformation. Gain of Function (GOF) diseases arise from the aggregation of a misfolded or partially folded protein. (Figure adapted and modified from references [3 and 4]).In loss of function diseases, the misfolded protein is unable to acquire its functional conformation, or to move to its site of action, or it is not recognized within the cell. These diseases are due to inherited or somatic mutations and include cystic fibrosis, lysosomal storage diseases (Gaucher’s and Fabry’s disease), α1-antitrypsin deficiency, and a variety of cancers [3]. GOF diseases are often associated to the accumulation of protein aggregates of the misfolded protein that can adversely affect cell function. Several age-related neuro-degenerative diseases (Alzheimer’s and Parkinson’s diseases), ALS, and amyloidoses belong to this class of diseases [4].
Protein Folding
The folding of a protein proceeds through an universal mechanism through which a polypeptide chain wraps itself into the structure that is most stable under physiological conditions. The Anfinsen’s experiment revealed that small, denatured proteins refold spontaneously in vitro [5], demonstrating that the information needed for proteins folding is implicit in the amino acid sequence. The free energy of a protein is affected by the following contributions: (1) the hydrophobic effect, (2) the energy of hydrogen bonds, (3) the energy of electrostatic interactions, and (4) the conformational entropy due to the restricted motion of the main chain and the side chains [6]. In 1969, Levinthal suggested that proteins’ folding is not due to a random search and specific folding pathways may exist [7]. This concept motivated a great number of researches designed to find and to describe the “folding intermediates”. These researches furnished a number of models that describe the folding process [8, 9]. Indeed, this process is highly complex and heterogeneous due to the very large number of potential conformations that can be adopted by a protein chain. To date considerable progress has been made in understanding this process. In the landscape theory, polypeptide chains have thus an energy landscape through which they can fluctuate during the folding reaction, which in this perspective can be likened to a funnel, moving along several downhill routes towards the native structure [10]. This quickly increases the number of protein interactions that speedily limits the conformational space that needs to be explored increasing the rate of the folding process. However, the free-energy surface is not smooth and proteins may be become trapped in a local minimum until it acquired sufficient thermal energy to surmount kinetic barriers and continue the folding process. The propensity of proteins to adopt a partially folded conformation increases in the presence of larger, complex domains that are stabilized by long-range interactions. Partially folded or misfolded states generally expose hydrophobic regions (amino-acid side-chain and/or polypeptide backbone) to the solvent so they consequently may aggregate in a concentration-dependent manner [10]. Protein aggregation may develop either amorphous structures or stable, highly ordered structures, named amyloid. These are characterized by the presence of β-strands perpendicularly to the fibril axis (cross-β structure). In vivo protein aggregation is controlled by the chaperone machinery, consequently it is more widespread under stress conditions or when the protein quality control system fails. The growth of protein aggregates is preceded by the formation of less ordered and rather heterogeneous soluble oligomeric proteins that represent the toxic forms of fibrillar protein. The toxicity of these molecules is strictly correlated with the presence of hydrophobic surfaces exposed to the solvent and not integrated into amyloid fibrils [11].
In the cell, protein folding is much more challenging than in a test tube owing to the highly crowded environment containing up to 200−300 mg/mL of proteins. In this context, proper protein folding, proteome integrity, and protein homeostasis depend on a complex network of protein chaperones, belonging to the protein quality control systems, which interact and stabilize unfolded proteins to reach their native conformation [12, 13]. These proteins recognize the presence of hydrophobic surfaces exposed to the environment and act shielding these surfaces. In this way, they suppress the protein aggregation and promote the protein folding. In addition to protein chaperones, the protein quality control systems in eukaryotic cells include proteases and accessory factors that work together to refold or remove misfolded proteins. Indeed, two different and complementary activities characterize this system. One controls the folding/refolding processes, protecting protein intermediates from aggregation, while the other eliminates proteins unable to correctly fold to the native state. To the proteostasis network belong the two stress-inducible pathways: the Unfolded Protein Response (UPR) and the Heat Shock Response (HSR).
Defects in proteostasis are detected by the UPR pathway that reduces the rate of the protein synthesis, increasing the synthesis of enzymes involved in the protein folding as well as those associated with the protein degradation machinery present in the endoplasmic reticulum and with the secretory activity. However, if proteostasis is not restored, UPR activates the death/senescence programs of cells. The cytosolic proteins belonging to the HSR system are controlled by the heat shock factors (HSF) proteins. Many of these proteins act as molecular chaperones that pilot the structure of proteins over protein folding processes, preventing misfolding, and aggregation. A prominent role is carried out by the HSF-1 protein. Its activity is essential in maintaining cellular proteostasis by regulating molecular chaperone expression. Several molecules activate the expression and/or activity of HSF-1 and other heat shock proteins. The induction of these proteins prevents protein damage and restores the cell to the pre-stress condition [14, 15].
SMALL MOLECULES
An emerging strategy for the treatment of diseases associated to protein misfolding is represented by the use of small molecules that act modulating the protein folding. These compounds are attractive because they can be administered orally and have a good potential to access to most cell types. Moreover, these molecules are advantageous in that they can cross the blood brain barrier, do not cause autoimmune responses, and have a low manufacturing cost [12, 16]. Low molecular weight compounds can be split into two categories: i) those that operate directly on the target protein acting either as pharmacological or chemical chaperones and ii) those that act indirectly, regulating either degradative pathways or protein folding catalysts.
In this chapter, the possibility of low molecular weight compounds to alter protein misfolding and restore the native conformation, acting either chemical or pharmacological chaperones, will be analyzed and discussed.
Small Molecules that Act as Pharmacological Chaperones
A pharmacological chaperone is a small molecule “whose function is to assist a protein to fold properly and enter its normal processing pathway smoothly” [17]. The finding that a ligand can bind to the native state of the protein promoting the native state and increasing the content of folded protein was a landmark discovery. Characteristics of pharmacological chaperones are their ability to target specific proteins and to be effective at low concentration, having so a reduced toxicity. Examples of pharmacological chaperones are compounds that interact with the active site of an enzyme acting either as substrates or as inhibitors. Nevertheless, any compound that specifically binds to the native state of a protein, also targeting different binding sites, may act as a pharmacological chaperone.
Specific limitations in the use of pharmacological chaperones may be represented by different factors. Firstly, the ligand must bind to the native state of the protein with an affinity sufficient to surmount the destabilizing effects of protein mutation. Secondly, if the ligand is also an inhibitor of the protein activity, its binding must be reversible and its active concentration as pharmacological chaperone must be lower than the inhibitory concentration. In this case, it is fundamental that the native state of the protein is stable when formed. Thirdly, the partially folded protein must adopt a conformation that recognizes and binds the pharmacological chaperone. Finally, protein mutations cannot involve neither the binding site nor sites required for efficient folding interactions. Despite these limitations, the effects of pharmacological chaperones on the control and correction of protein misfolding are very exciting. To date several applications of pharmacological chaperones have been reported showing these molecules to be especially effective in the treatment of different diseases associated either to loss of function or to amyloid aggregates accumulation (GOF diseases).
Lipid Storage Disorders
Lipid storage disorders belong to the family of the rare inherited disorders of lysosome function classified into more than 40 pathologies [18]. Lysosomal enzymes are synthesized in the rough endoplasmic reticulum and then are delivered to organelles by the endosome-lysosome pathways. In the most known lipid storage disorders, the Gaucher’s and Fabry’s diseases, the use of pharmacological chaperones has been reported to alleviate their progression, restoring the native folding of the enzymes associated to these pathology.
Fabry’s disease is a metabolic disorder involving a deficiency in the lysosomal α-galactosidase A (α-Gal A), an enzyme that hydrolyzes the terminal α-galactosyl moieties from glycolipids and glycoproteins. Fan and colleagues [17] found that a ceramide analog, the 1-deoxy-galactonojirimycin (DGJ) (1, Fig. 2), that acts as a competitive inhibitor of α-Gal A, showed properties that were not at all attributable to its inhibitory activity. Using lymphoblasts containing enzyme mutations associated with a late-onset form of the Fabry’s disease that affects the heart (R301Q or Q279E), the authors found that in presence of a sub-inhibitory concentration of DGJ (20 μM) the activity of α-Gal A increased 8- to 10-fold, and more important, the effect persisted for several days after withdrawal of the drug. Surprisingly, using DGJ concentrations greater than 20 μM the enzyme activity did not increase. Further experiments revealed that the mutant enzyme localized into the Golgi apparatus, whereas in cells treated with DGJ, the mutant protein localized to the lysosome. Moreover, the interaction of DGJ with purified mutant enzyme inhibited its unfolding at pH 7. Overall, these data indicated that DGJ at sub-inhibitory concentrations acts as a pharmacological chaperone promoting the folding and the trafficking of mutant α-Gal A through the endoplasmic reticulum to lysosome.
Fig. (2)) Chemical structure of 1-deoxy-galactonojirimycin derivatives.The binding of DGJ to the active site of mutant α-Gal A is much tighter at pH 7.0 (endoplasmic reticulum pH) than at pH 4.5 (lysosomal pH). The tight binding of DGJ to α-Gal A is thought to induce the folding of the mutant enzyme, and successively, the folded enzyme–DGJ complex successfully transits to the lysosome. Once in the lysosome, the complex dissociates and the mutant enzyme maintains its structure, displaying the normal enzymatic activity. This is possible because the mutations cause the misfolding of α-galactosidase A at pH 7.0 but not in the lysosomal environment (pH 4.5).
Similarly, to the DGJ-enzyme interaction, also the binding of the N-butyl -derivative (NB-DGJ, 2) to acid β-glucosidase was found to be pH-dependent [19]. The result showed that at neutral pH NB-DGJ binds to enzyme, while at middle acidic lysosomal conditions it does not bind to them. The N-butyl as well as the N-nonyl derivative (NN-DNJ, 3) are potential pharmacological chaperones to treat adults with Gaucher’s disease. This was recently confirmed for the disorder characterized by the N370S mutation, the most prevalent mutation associated with this pathology. Moreover, molecules 2 and 3 act also as inhibitors of the glucosyl synthase and acid β-glucosidase (Fig. 3), respectively, two enzymes involved in this disease.
Fig. (3)) Acid β-glucosidase catalysed reaction. Mutations of this enzyme cause an accumulation of glucosylceramide in lysosome characteristic of the gaucher’s disease.Sawkar et al. [20] demonstrated that incubating patient-derived fibroblasts for nine days with a sub-inhibitory concentration of 3 (10 μM) led to a twofold increase in the activity of mutated acid β-glucosidase. Moreover, the increased activity persisted for at least 6 days after the withdrawal of the drug. Experiments with purified wild-type acid-β-glucosidase revealed that NN-DNJ protected the enzyme in a dose-dependent manner from heat denaturation. These results suggested that NN-DNJ acts as a pharmacological chaperone with a mechanism very similar to that described for DNJ, promoting the folding and the trafficking of the mutant enzyme through the endoplasmic reticulum to the lysosome, where it displays normal enzymatic activity.
Amyloid Related Diseases
Amyloidoses are slow and progressive diseases that result from a gain of function of unfolded form of different proteins. Neuropathic (e.g. Alzheimer’s, Parkinson’s, Alexander’s, and others diseases) and non-neuropathic diseases (e.g. type II diabetes and various forms of systemic amyloidosis) are characterized by the presence of amyloid deposits in the brain or in different organs, respectively. More than 30 different peptides and proteins (Aβ-peptide, tau, α-synuclein, huntingtin, amylin, β2-microglobulin, lysozyme, etc.) were discovered to be able to form amyloid fibrils. The formation of amyloid aggregates begins with the destabilization of the native conformation of protein that successively rearranges through the formation of new non-covalent intermolecular interactions. The unrelated functions, as well as the different amino acid sequences, size, and structures of amyloidogenic proteins/peptides suggest that the ability to form amyloid fibrils is a property of the polypeptide chains, although this process is strongly favorite either by mutation of the native proteins or by environmental factors (high protein concentration, metal ions, and others).
A wide range of molecules has been tested as inhibitors of amyloid aggregation and/or promoter of disaggregation of amyloid fibrils in in vitro systems. Among these, the most studied are natural polyphenols extracted from plants. Interestingly, also approved drugs for the treatment of non-protein misfolding-related diseases have showed to possess antiamyloidogenic activity, offering the possibility of new life for different compounds.
Transthyretin (TTR), a serum protein that is the main transport of retinol associated with the retinol binding protein [21], also binds the secondary thyroid hormone thyroxine (4, Fig. 4). This protein adopts a homotetrameric structure of four identical subunits comprising 127 amino acids with two thyroxine-binding sites in the central groove. Different human disorders related to more than 80 mutations in the gene encoding the TTR subunits are associated with the deposit of TTR fibrils in various organs, including the nerves, heart, and kidneys. Mutant TTR deposits lead to familial amyloid cardiomyopathy (V122I), familial amyloid polyneuropathy (V30M), and central nervous system amyloidoses (D18G and A25T). Moreover, also wild-type TTR itself is amyloidogenic and its aggregation is related with the senile systemic amyloidosis, a cardiomyopathy that involves up to 20% of the population over age 65 [22].
The rate-limiting step in TTR aggregation is the dissociation of TTR tetramer. TTR tetramer dissociates in two dimers that then dissociates to unstable monomers that easily unfold and aggregate. It is found that small molecules, known as kinetic stabilizers, able to bind to TTR may stabilize the tetramer structure and counteract the TTR aggregation.
Fig. (4)) Chemical structure of transthyretin kinetic stabilizer.Miroy et al. [25] demonstrated that thyroxine (4) and its analogue 2,4,6-triiodophenol (5) interact with the TTR tetramer and inhibit amyloid formation, confirming this hypothesis. The structural information on the thyroxine-TTR complex (Fig. 5) permitted to design over 1000 aromatic small molecules that stabilize the TTR tetramer. This work found that diflunisal (6), an FDA approved non-steroid anti-inflammatory drug, and tafamidis (7) interact with TTR, stabilizing the tetramer form [26, 27]. Unfortunately, diflunisal, currently in phase III clinical trial to evaluate its effectiveness in the treatment of familial amyloid polyneuropathy, familial amyloid cardiomyopathy, and senile systemic amyloidosis, shows a modest selectivity for TTR. On the contrary, tafamidis binds to TTR with an excellent selectivity over other plasma proteins.
A new promising approach to counteract TTR aggregation is represented by the use of bivalent ligands [28]. Characteristic of these molecules is the ability to bind simultaneously to both the thyroxine-binding sites of the TTR tetramer. The crystal structure of the complex of TTR with the ligand 8 [29] shows that an alkyl chain of 7-10 Å is suitable to fit comfortably two biaryl ligands into both the thyroxine binding sites of the tetramer.
Fig. (5)) Structure of human transthyretin complexes with thyroxine [23]. The structure was made from X-ray coordinates (PDB code 2ROX) using the PyMOL software package [24].Hereditary systemic amyloidosis is a disease related to a single mutation in the lysozyme gene [30]. This enzyme is a widely distributed protein able to hydrolyze the β-1,4 glycosidic linkages between the N-acetylmuramic acid and N-acetylglucosamine groups in the peptidoglycan cell wall structure of Gram-positive bacteria. Human lysozyme, as well as hen egg white lysozyme, is a commonly used model system of protein structure and function, as well as to study the mechanisms of protein folding stability [31]. In patients affect by hereditary systemic amyloidosis massive amyloid deposits are present in different organs, particularly in the liver and kidneys, in the connective tissue and in the walls of blood vessels. To inhibit the lysozyme aggregation Gazova et al. [32] have considered the use of pharmacological chaperones. The authors investigated the ability of acridine-based compounds, reported as defribillization molecules, to bind to misfolded lysozyme and act as scaffold for the misfolded enzyme. Three different acridine groups, classified according their chemical structures (planar-, spiro-, and tetrahydro-acridines) were screened as anti-aggregating compounds. Only planar acridines were found to be able to inhibit lysozyme aggregation (compounds 9-16, Fig. 6), while spiro- and tetrahydroacridines did not interfere with lysozyme fibrillization. The two most potent compounds, 10 and 12, have an IC50 value less than 10 μM. The activity of planar acridines may be explained by the possibility of the flat heterocyclic skeleton to intercalate between the hydrophobic protein surfaces, disrupting the β-sheets structure.
Fig. (6)) Chemical structures of planar acridine derivatives [32].Recently, the capability of plant polyphenol to inhibit lysozyme self-aggregation as well as to dissolve its amyloid aggregates has been explored [33]. The results demonstrated that quercetin (17, Fig. 7), resveratrol (18), and caffeic acid (19) are more efficient to depolymerize amyloid aggregates than to inhibit their formation. The authors also found that in vitro these molecules act in a synergistic manner.
Fig. (7)) Chemical structures of plant polyphenols [33].The potential therapeutic role of polyphenol compounds, in particular of curcumin (20, Fig. 8) and curcumin-like molecules, has also been explored in neurological disorders including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Curcumin, a polyphenol extracted from turmeric, a commonly used spice in Asia cuisine, also possess anti-inflammatory and anti-oxidant activities that improve its neuroprotective activity [34]. Recent studies found that curcumin binds to amyloid Aβ oligomers and/or fibrils, altering its aggregation, and reducing the toxicity of fibrils in cell model of Alzheimer’s disease. Curcumin also inhibits the in vitro α-synuclein aggregation, attenuating the toxicity of α-synuclein oligomers in cells.
Fig. (8)) Degradation by-products of curcumin.In a recent work, Singh et al. [35] found that in vitro curcumin preferentially binds to the preformed α-synuclein oligomers, accelerating their aggregation and consequently, reducing the soluble cytotoxic oligomers. Unfortunately, curcumin is low soluble in aqueous solution and it is rapidly degraded at physiological pH into ferulic acid, vanillin, and dehydrozingerone (Fig. 8). The capability of dehydrozingerone (21, Fig. 9), its O-methyl derivative (22), zingerone (23), and their C-2 symmetric dimers (biphenyls 24-26, respectively) to interact with α-synuclein and to modulate its aggregation process have been explored [36]. The results showed that biphenyl analogues 24 and 25 interacted with α-synuclein inhibiting its aggregation more efficiently that the corresponding monomer compounds.
Fig. (9)) Chemical structure of curcumin-like molecules and their biphenyl derivatives [36].In 2010, Sechi at al. [37] demonstrated that the β-lactam antibiotic ceftriaxone (27, Fig. 10), a safe and multi-potent agent used for decades as antimicrobial, was able to eliminate the cytotoxic effects of misfolded glial fibrillary acidic protein (GFAP), in an in vitro model of Alexander’s disease (AxD). Pathogenetic determinants of this disease include a GOF of mutated GFAP that causes the growth of protein aggregates in astrocytes, containing mutant GFAP, and other proteins. The authors reported that a 20-month course of intravenous, cyclical ceftriaxone administration in a patient with an adult form of AxD induces a successful clinical course. A four-year-long extension of the trial in this patient confirmed that ceftriaxone halted and reversed the progression of neuro-degeneration, and a significant improvement of the quality of life of the patient has been reported [38]. Recently, the ability of ceftriaxone to ameliorate motor deficits in a rat model of Parkinson’s disease has been recognized [39]. In vitro experiments demonstrated that ceftriaxone bind to α-synuclein with good affinity, inhibiting its in vitro aggregation. Moreover, ceftriaxone also protects PC12 cells against 6-OHDA-induced damage [40]. Collectively these data suggests that the treatment with this molecule may be an effective approach to treat neurodegenerative disorders characterized by the presence of amyloid fibrils.
Recently, some dyes such as methylene blue (28, Fig. 10) and Congo red (29) have been tested as inhibitors of the protein aggregation. Methylene blue is a phenothiazine dye approved by FDA for oral and i.v. administration for several pathologies. Its administration in Alzheimer patients slows down the progression of the disease with an improvement of cognitive functions [41], even if the actual mechanism of action of this drug is still debated. In vitro experiments show that methylene blue antagonizes the aggregation of Aβ42 peptide [42] and of other amyloidogenic proteins [43] including the prion protein [44]. Congo red is a dye traditionally used to detect the presence of amyloid fibrils in tissues through birefringence assay [45]. In solution, Congo red similarly to Lacmoid (30) associated in supramolecular structures [46 and reference therein]. Both these molecules, Congo red and Lacmoid, bind to monomeric α-synuclein, although to different region and with different affinities, affecting the fibril formation [47].
Fig. (10)) Chemical structure of small molecules interacting with either α-synuclein or Aβ peptide.Cystic Fibrosis
Cystic fibrosis is an autosomal recessive genetic disorder characterized by the presence of dense, viscous secretions due to an abnormal transport of chloride and sodium across the epithelium. The deletion of the Phe508 residue (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein is the most common cause of this disease [48]. This causes the misfolding of the full-length protein that is retained in the ER and degraded rather than trafficked to the plasma membrane. Different less frequent mutations of this protein are associated either to impairing channel function (G551D-CFTR) or with both the above reported aspects. To restore the function of mutated CFTR, small molecules with different activity (knowed as "correctors" and "potentiators") have been proposed. Molecules that act as “correctors” are able to overcome the defect process involving the ΔF508-CFTR mutant protein and transport it to cell surface, while molecules that acts as “potentiators” enhance ATP-dependent channel gating functions [49]. CFTR correctors, at the current time, must be identified by high-throughput screening because there is insufficient information to design rational drugs. These molecules may be either substrate or competitive inhibitors of CFTR [49]. Compound 31, named Lumacaftor, is an effective in vitro corrector of the ΔF508-CFTR folding (Fig. 11). At the moment, it enters in phase III trials in association with Ivacaftor (32) a molecule that act as CFTR potentiator [50].
Fig. (11)) Chemical structure of CFTR corrector (31) and potentiator (32), and of 4-phenylbutyric acid (33).In 1997, Rubenstein et al. [51] found that 4-phenylbutyric acid (33), an FDA approved drug as ammonia scavengers in urea cyclic disorders, was able to increase the trafficking of ΔF508-CFTR to the cell surface acting as chemical chaperone [52]. In these years, the chaperone-like activity of 33 have expanded significantly in different protein misfolding diseases preventing the accumulation of unfolded and aggregated protein [53 and references therein], e.g., the mutant form of the protease inhibitor alpha-1 anti trypsin, or α-synuclein.
Small Molecules that Act as Osmolytes
Chemical chaperones are low molecular mass molecules able to stabilize the protein structure against thermal or chemical denaturation, and most important, might overcome folding defects [54]. The use of chemical chaperones as protein folding agents is well documented; unfortunately, they are effective at high concentration, at least micromolar level, through weak thermodynamic interaction with substrate. An example of chemical chaperones is represented by osmolytes. Contrary to inorganic ions, osmolytes help cells to counteract extracellular stress [55], not interfering with structure or function of biological macromolecules. For instance, the addition of either KCl or NaCl disrupted the native myofilament architecture that is precluded by the addition of trimethyl amine oxide (41, Fig. 12). Similarly, in the presence of inorganic ions the enzymatic activity of pyruvate kinase of the crab is strongly reduced, while the addition of osmolytes does not affect it. Osmolytes can be grouped into four chemical classes (Fig. 12): sugars (34 and 35), polyalcohols (36 and 37), amino acids and their derivatives (38 and 39), and methyl ammonium compounds (40 and 41).
Osmolytes occur at very high concentrations in cells (0.1–1 M), they do not bind to proteins, and they have a unique ability to stabilize native folded proteins, destabilizing potentially toxic unfolded proteins, in response to rapid changes in the external and internal environment. The mechanism proposed to explain the effect of osmolytes to stabilize proteins from denaturation was based on an exclusion phenomenon: osmolytes stabilize the native state of proteins because of the dramatic destabilization of the unfolded state [56]. Osmolytes have an unfavorable interaction with the surface of a native protein and an even less favorable interaction with the peptide backbone of the denatured/unfolded protein. The inability of an osmolyte to interact with the surface of a native protein in aqueous solution increases the free energy of the native state in contrast to the free energy of the native state in the absence of osmolyte. Since in the unfolded state there is far more surface area than in the folded state, and since osmolytes fail to interact with the peptide backbone, the free energy of the unfolded state of a protein in an aqueous solution of osmolyte is much larger than the free energy of the unfolded state in the absence of osmolyte. Thus, osmolytes stabilize the globular structure of proteins by favoring compaction. Consequently, the addition of osmolytes, in the case of intrinsically disordered proteins may be a disadvantage. Indeed, compaction can promote protein aggregation. Additionally, osmolytes show some chemical activities such as antioxidant (polyols and taurine), redox balance (glycerol), detoxifying sulfide (hypotaurine), and membrane stabilization under freezing temperatures (trehalose) [55].
Fig. (12)) Chemical structures of some osmolytes.Trehalose
Trehalose (35) is a disaccharide of glucose present in many non-mammalian species that prevents protein denaturation protecting cells against various environmental stresses. Tanaka et al. [57] demonstrated the potential of trehalose for treating Huntington’s disease, a fatal poly-glutamine (polyQ) GOF disorder that causes neurodegeneration in humans. Like other polyQ diseases, age of onset and severity are inversely related to the length of the polyQ segment attached to the N-terminus of the huntingtin protein. The polyQ sequence promotes the aggregation of protein, killing the cells, although the mechanism of cell death remains unknown. An elegant screen to test compounds able to disrupt polyQ aggregates has been developed by these authors. Myoglobin, a protein that does not aggregate at 37°C, was engineered by the addition of a polyQ sequence (35 Gln residues) to its N-terminus. The new protein readily aggregates at 37°C and the process is easily monitored by following the absorbance at 550 nm. The screening of about 200 compounds showed that disaccharides in general, have a propensity to reduce protein aggregation, and that the best inhibitors are trehalose and a tetramer of N-acetylgalactosamine.
The capability of trehalose to interfere with protein misfolding and aggregation has been evaluated also in other gain of function diseases. Liu et al. [58] analyzed the ability of this disaccharide to inhibit in vitro the aggregation of Aβ40 and Aβ42 peptides as well as to dissolve their preformed aggregates. These peptides are two cleavage products of the amyloid beta protein (Aβ) particularly prone to aggregate, involved in the composition of amyloid fibrils characteristic of the Alzheimer’s disease. The authors found that a low concentration of trehalose (< 50 mM) completely inhibited the aggregation of Aβ40 and significantly dissolved its preformed aggregates, while only partially inhibited (50%) the aggregation of the more toxic peptide Aβ42. Cytotoxic assay showed that preformed aggregates of Aβ40 co-incubated with 50 mM trehalose were not toxic to human neuroblastoma SH-SY5Y cells, confirming the neuroprotective activity of this molecule.
Jiang et al. [59] explored the capability of trehalose to control the amyloidogenic properties of α-synuclein, an intrinsically disordered protein involved in different diseases (synucleinopathies), including the Parkinson’s disease. In pathological conditions, this protein forms insoluble fibrils and aggregates (named Lewy’s bodies). The authors found that low concentration of trehalose disaggregates preformed mutated α-synuclein (A53T) protofibrils and fibrils into small aggregates or even random coil structure. Increasing the concentration of trehalose, the authors observed that the transition into β-sheet structure is slowed down and the formation of mature A53T α-synuclein fibrils is completely prevented. In vitro experiments revealed that α-synuclein co-incubated with trehalose assembles into large amorphous aggregates rather than to neurotoxic fibrils, and that long time of incubation with trehalose disassembled large amorphous aggregates up to the random coil structure. In addition, in vitro experiments in transduced PC12 cells showed that trehalose at a concentration lower than 1.0 mM inhibited the overexpression of α-synuclein and protected the cells from the toxicity of α-synuclein fibrils [59].
Recent data indicate that the in vivo activity of trehalose may be somewhat more complicate. Indeed, it appears to be also an inducer of autophagy [60], a cellular process that serves to rid cells of protein, protein aggregates, and even whole organelles by the transfer to lysosome. In this way, trehalose further contributes to the elimination of protein aggregates.
Trimethylamine N-Oxide
Trimethylamine N-oxide (TMAO) (41) is a “counteracting” osmolyte as betaine and glycerophosphocholine that are naturally accumulated in mammalian kidney. On the contrary of sucrose and some amino acids, classified as compatible osmolytes, that affect only protein stability, counteracting osmolytes influence both protein stability and function. Experimental data show that TMAO increases both the melting temperature and the unfolding free energy of proteins, counteracting the denaturating effects of urea. Indeed, the treatment with TMAO restores the enzyme activity lost upon urea treatment. The chemical structure of TMAO suggests two different possible types of intermolecular interactions of this molecule with proteins. The unshared electron pair on the oxygen may act as a hydrogen bond acceptor forming hydrogen bonds with proteins. Alternatively, the methyl groups may be involved in hydrophobic interactions with amino acid side chains. In silico studies demonstrated that TMAO preferentially interacts with charged and polar amino acid side chains as well as with the protein backbone [61]. To analyze the action of TMAO at molecular level, Cho et al. simulated the effect of different TMAO concentration on the aggregation of the Aβ16-22 (KLVFFAE) monomer, showing that TMAO acts as a nano crowder that limits the degrees of freedom of the unfolded state and entropically destabilizes it [62].
Evaluation of small molecules effects on protein folding
Circular Dichroism (CD) Spectroscopy
CD spectroscopy is a quick and useful technique to explore the structure and the conformational stability of proteins and peptides in solution. CD experiments permit to determine whether a protein is folded, its secondary structure, and some aspects of its tertiary structure by analyzing either the far-UV (190-250 nm) or the near-UV (250-300 nm) region of the spectrum, respectively. In addition, CD measurements permit to investigate the capability of a ligand to interact with a protein, to determine its affinity and how the protein structure is affected. Additionally, CD is used to evaluate the protein stability in function of temperature, pH, and denaturant conditions. If the change in CD spectrum as a function of temperature is reversible, the data are used to determine the thermodynamic parameters of protein unfolding and the melting temperature (TM) that corresponds to the midpoint of the unfolding transition curve. The melting temperature reflects the global stability of the folded versus the unfolded protein. For example, in the presence of 0.4 M glycerol, myo-inositol and trehalose the TM value of malate dehydrogenase increased by 3 to 5°C in comparison to the protein alone [63]. On the other hand, melting experiments performed in the presence of osmolytes indicated that proteins conserved an important content of secondary structure at the TM value of the proteins alone [63]. Studies on RNase A showed that the addition of trehalose increases the ΔHm value lowering the ΔCp-value (heat capacity change) [64]. The ΔCp is a thermodynamic parameter that associates the exposure of the protein surface to the solvent [65]. A decrease in ΔCp is associated to a lower exposed surface area and/or a decreased exposure of hydrophobic side chains to the solvent that may result in flattening of thermal unfolding curves.
Recently Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy has been used to study protein structure and protein-ligand interactions. In the SRCD spectroscopy, the high intensity synchrotron radiation is used as light source. The high-flux beam has a number of important spectral consequences for CD studies. Among these, the acquisition of data at very low wavelength UV containing additional information on secondary structures, the higher signal-to-noise ratios, the decreased requirements for signal averaging and the possibility to collect CD spectra in the presence of buffers and absorbing components mimicking physiological conditions [66, 67]. An undesirable effect observable using higher-energy synchrotron is the protein denaturation induced by the higher photon flux of the synchrotron source. Indeed, a significant decrease of secondary structure upon light irradiation using intense far-UV radiations is well observable in structured proteins with significant content of α-helical and/or β-sheet conformations [68]. Recently, UV denaturation experiments have been proposed to evaluate the effects of small molecules on protein stability [69]. Indeed, UV denaturation, like thermal denaturation, varies from protein to protein and, for the same protein, by the presence of ligands. This effect can be used to evaluate protein stability, and how the protein stability is modulated by the presence of ligands or osmolytes. UV denaturation, initially attributed to the effect of free radicals that may be generated by the high photon flux, was postulated to be due to the heating of internal bound water molecules involved in maintaining the protein native structure. As show in Fig. (13), the effect of ceftriaxone on GFAP protein structure has been evaluated by SRCD spectroscopy. The addition of ceftriaxone increases the α-helical content of the protein as well as its stability towards UV denaturation confirming the protective role of ceftriaxone [70].
Fig. (13)) (A) Far-UV SRCD spectra of GFAP alone (0.66 mg/ml) in aqueous solution at different time of irradiation. (B) UV-protein denaturation curves of GFAP alone and in presence of ceftriaxone (Protein/ Ligand Ratio was 1:2). The normalized CD signal at 190 nm was reported vs. experiment number (irradiation time). GFAP concentration was 0.66 mg/ml; ceftriaxone concentration was 270 µM [70].Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR spectroscopy, permitting to characterize proteins at the level of single amino acid residues, has significantly contributed to comprehending the mechanism of protein folding. NMR techniques used to monitor the effects of temperature, pH, denaturants, and solvent on protein conformation and folding are either 2D 1H correlation experiments or Heteronuclear Single Quantum Coherence (HSQC) experiments with 15N-labeled or/and 13C-labelled proteins that permit to evaluate the chemical shift modifications due to ligand interaction. HSQC experiments also permit to localize the ligand binding sites on assigned protein. Limits in this approach are the size of the protein analyzable by NMR, the need to have milligram quantities of 15N- or/and 13C-labelled pure, soluble protein, which should be stable throughout the battery of multi resonance experiments and the time consuming process of resonance assignments. For these reasons, target-based detection methods are normally used for proteins with solved structures, while less time-consuming ligand-based detection approach is generally used for novel protein-ligand binding characterization studies.
Molecules in a solution exhibit random translational motion (Brownian motion) by virtue of their internal kinetic energy. Self-diffusion of a molecule depends on its size and shape in solution, and is modified upon binding to a macromolecule. This makes the determination of self-diffusion coefficients an important methodology for molecular interaction studies in solution. Pulsed field gradient NMR diffusion experiments are one of such powerful techniques that is used to measure the translational diffusion of both small molecules and macromolecules and for mapping ligand-protein interactions. It is a non-invasive, cheap, and easymethod that can obtain physical, chemical, and qualitative information about the components in the sample mixture [71, 72].
An emerging technique to analyze internal dynamics of proteins is represented by both heteronuclear NMR relaxation and relaxation dispersion experiments. The measurement of parameters related to the N-H bond vector motion such as the longitudinal (T1) and transverse (T2) relaxation rate and the heteronuclear NOE (Nuclear Overhauser Effect) yield useful information about protein dynamics. Relaxation data (T1, T2, and NOE) are correlated to an order parameter (S2) that varies from 0 (no motional restriction) to 1 (complete motional restriction) [73]. Flexible backbone segments have low S2 values, whereas rigid regions have high S2 values. Upon the addition of osmolytes, flexible backbone segments become more rigid and the overall protein compactness increases, consequently the S2 value reported per residue basis increased.
NMR relaxation dispersion experiments allow to characterize the conformational change between the native ground state and partially folded states that generally are not detectable in the NMR spectra (low populated and short-lived) [74, 75].
OUTLOOK
This chapter examined the role that pharmacological and chemical chaperones play in protecting cells from a loss or gain of protein functions related to protein misfolding. It is evident that understanding of how mutations and/or intracellular factors affect protein folding and how small molecules (chemical or pharmacological chaperones) may restore the natively folded protein requires the knowledge of both wild-type and mutant protein structures. Gaps remain in comprehending folding intermediates and the conformational changes correlated with the transition from native to misfolded states. The use of chemical chaperones may led to understand how misfolded protein can be converted to functional folded protein accelerating the finding of therapeutic pharmacological chaperones. At the molecular level, the kind of molecule required to overcome a specific disorder is strictly related to the structure of a protein and how its misfolding leads to either loss or gain of functions. Despite the challenge mentioned in this chapter, the possibility of controlling proteostasis by the use of small molecule is notable and continues to advance. However, a careful consideration requires, to be assumed regarding the specificity of small molecules and their potential effects on the modulation of other cellular processes.
CONFLICT OF INTEREST
The author (editor) declares no conflict of interest, financial or otherwise.
Acknowledgements
We thank Diamond Light Source for access to beamline B23 (SM8034-1) that contributed to the results presented here. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 226716.
