A New Face for Old Antibiotics: Tetracyclines in Treatment of

Perspective pubs.acs.org/jmc

A New Face for Old Antibiotics: Tetracyclines in Treatment of Amyloidoses Tatiana Stoilova,† Laura Colombo,† Gianluigi Forloni,† Fabrizio Tagliavini,‡ and Mario Salmona*,† †

IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Via Giuseppe La Masa 19, 20156 Milano, Italy Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Via Celoria 11, 20133 Milano, Italy

‡

ABSTRACT: The use of tetracyclines has declined because of the appearance of resistant bacterial strains. However, the indications of nonantimicrobial activities of these drugs have considerably raised interest and triggered clinical trials for a number of different pathologies. About 10 years ago we first reported that tetracyclines inhibited the aggregation of prion protein fragments and Alzheimer’s β peptides, destabilizing their aggregates and promoting their degradation by proteases. On the basis of these observations, the antiamyloidogenic effects of tetracyclines on a variety of amyloidogenic proteins were studied and confirmed by independent research groups. In this review we comment on the data available on their antiamyloidogenic activity in preclinical and clinical studies. We also put forward that the beneficial effects of these drugs are a result of a peculiar pleiotropic action, comprising their interaction with oligomers and disruption of fibrils, as well as their antioxidant, anti-inflammatory, antiapoptotic, and matrix metalloproteinase inhibitory activities.

I. INTRODUCTION

ability to move and function independently and ultimately leaving them vulnerable to fatal infections. Amyloidogenic proteins fold independently of their primary structure and molecular weight. This stands in contrast to natively folded proteins that fold to a specific three-dimensional structure strongly dependent on their amino acid sequence, while amyloidogenic proteins under certain conditions can aggregate and form fibrils with a very similar ultrastructure and properties.4 In tissue deposits all amyloid fibrils present themselves as rigid, nonbranching structures of approximately 10 nm in diameter, which bind the dye Congo red and exhibit green birefringence when viewed under cross-polarized light.2 In addition to Congo red, which specifically binds to β-sheet rich structure resulting in characteristic red shift in the absorption band of the dye, thioflavin T (ThT) is also widely used for the identification of amyloid fibrils. This dye fluoresces strongly when it is added to samples containing β-sheet rich structure. When isolated and analyzed by X-ray diffraction, amyloid fibrils demonstrate a typical cross-β diffraction pattern with two characteristic reflections: a relatively sharp and

The term amyloidosis comprises more than 25 unrelated human pathologies, all of which are characterized by extensive misfolding and assembly of normally soluble proteins followed by inappropriate deposition of fibrils that disrupt the structure and function of tissues and organs.1,2 These diseases include both systemic amyloidoses in which protein aggregates may occur in any part of the body and localized amyloidoses in which deposits are presented in a single organ. Systemic amyloidosis is usually fatal and causes about 1 death per 1000 individuals in developed countries.3 To date, 14 human amyloid proteins with respective precursors are known to cause systemic amyloidoses. 2 These include, but are not limited to, immunoglobulin light chain (LC), serum AA, β2-microglobulin (β2M), transthyretin (TTR), apolipoprotein A-I, apolipoprotein A-II, gelsolin, and lysozyme amyloid proteins.2 The most common representatives of localized amyloidoses are Alzheimer’s, Parkinson’s, and prion protein diseases, characterized by accumulation and deposition of amyloid β (Aβ), αsynuclein, and prion protein (PrP), respectively. These pathologies affect the nervous system and cause dementia and/or motor disturbances, gradually destroying a person’s © XXXX American Chemical Society

Received: January 31, 2013

A

dx.doi.org/10.1021/jm400161p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Chemical structures of eprodisate disodium (1), tafamidis (2), 4′-iodo-4′-deoxydoxorubicin (3), and rifampin (4).

intensive meridional reflection at 4.7−4.8 Å and a more diffuse signal on the equator at approximately 10 Å, corresponding to the β-strand and β-sheet distances, respectively.2,4,5 The meridional reflection, which is derived from the fairly constant distance between hydrogen bonded β-chains, is highly conserved among all amyloidogenic proteins. This indicates a very similar core structure of the fibrils. By contrast, the equatorial reflection, which comes from the polypeptide sequence and side chain dependent distance between β-plated sheets, can vary from 8.8 to 14.6 Å.6 Amyloid fibrils, a principal component of plaques, were regarded for many years as the primary cause of cellular degeneration and pathogenesis of amyloidoses.7 However, a number of studies strongly suggest that cytotoxicity and initiation of degenerative cascades are rather caused by the small soluble oligomeric species that precede fibril formation.8−10 This can explain the little to no correlation between the density of amyloid deposits and disease manifestation observed in Alzheimer’s and prion diseases, as well as in type 2 diabetes mellitus and familial amyloid polyneuropathy (FAP).11−14 In fact, abundant amyloid deposits that characterize Alzheimer’s disease (AD) were detected in some cognitively normal individuals who did not show any neurodegenerative symptoms.15 A strong correlation was observed, however, between soluble amyloid β oligomer levels and the extent of synaptic loss and severity of cognitive impairment.13 Similarly, islet amyloid deposits associated with type 2 diabetes have been detected in nondiabetic humans. In transgenic mice carrying human amylin/islet amyloid polypeptide genes, the load of fibrillar amyloid did not correlate with their lifespan.16,17 This has led some researchers to hypothesize that the sequestration of misfolded protein in final amyloid fibrils and plaques has a protective function.18 However, formation of large amyloid deposits that impair the physiological functioning of cells and tissues can hardly be considered beneficial in patients affected by amyloidoses. Thus, it was recently shown that fibrillar β2M was cytotoxic to monocytes and chondrocytes and impaired osteoclast formation and viability. The result was decreased resorption of old bones, altered cartilage formation, and bone remodeling.19 Lipid membrane disruption caused by β2M fibrils and leakage of toxic species from mature fibrils were also described.20

To date, while the exact mechanism behind cellular degeneration remains unclear, several potential mechanisms of toxicity have been proposed. They were summarized in a review18 as follows: (1) tissue architecture destruction and organ dysfunction caused by deposition of large amounts of fibrils;21 (2) permeabilization and destabilization of mitochondrial and plasma membrane by oligomeric species;22 (3) oxidative damage produced by reactive oxygen species caused by the incorporation of redox metals into amyloids;23 (4) general disorganization of cellular protein homeostasis essential for cell viability by sequestration of numerous proteins, mostly metastable ones, in a “chaperone-like” manner.24,25 Virok et al. have used a protein array approach to discover Aβ interaction patterns, and more than 324 cellular proteins were identified as possible interactors with oligomeric forms of Aβ.24 Among them, 24 proteins were found to be involved in protein translation processes, suggesting that this might be the primary target of Aβ toxicity.24 In a recent paper, Manzoni et al. reported that Aβ bound to a variety of membrane proteins and, to a lesser extent, cytosolic proteins. This finding confirms the idea of the existence of multiple molecular targets of amyloid oligomers as opposed to a single specific receptor.25 Despite a great number of hypotheses, the exact mechanism of amyloid toxicity remains elusive. It is still unclear which pathway should be targeted for therapeutic intervention. The current search for disease-modifying therapies includes the following key strategies: (i) reduction of amyloid production; (ii) inhibition of amyloid aggregation and/or destabilization of aggregated species; (iii) promoting of its clearance.26 The first strategy, aimed at inhibition of amyloid formation, frequently produces a halt in disease progression. This strategy is based on decrease of the respective amyloid precursor protein production or its stabilization. For example, in serum A amyloidosis administration of anti-inflammatory and immunosuppressive drugs suppresses inflammation and decreases production of serum amyloid A protein, thereby slowing disease progression.1 In another type of systemic amyloidosis, the light-chain amyloidosis, the reduction of amyloid production is achieved by hematopoietic stem cell transplantation and administration of melphalan and dexamethasone, which are directed against the plasma cells responsible for the precursor protein synthesis.27 However, these drugs are often ineffective and poorly tolerated, producing a number of B



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Figure 2. Chemical structures of tetracyclines cited in the text (5−20). Inset 2A: General structure of tetracycline skeleton with atom numbering and ring labels of the hydronaphthacene carboxamide nucleus.

side effects like diminished production of blood cells, gastrointestinal problems, and increased risk of infection.27 In hereditary transthyretin amyloidosis, production of amyloidogenic mutant TTR in the liver is abolished by orthotopic liver transplantation.28 At the same time, it was found that in patients carrying some types of TTR mutation, liver transplantation may cause acceleration of amyloid cardiomyopathy due to the deposition of wild-type TTR fibrils in the heart.29 It is worth noting that amyloid fibrils isolated from patients after liver transplantation show a predominance of wild-type over mutant TTR, at variance with amyloid deposits from untreated patients.30 In patients with combined heart and liver transplantation, amyloid deposition in the heart did not occur. However, progression of amyloid formation in other tissues, such as peripheral nerves, kidneys, and adipose tissue, was observed.30 The cause of this side effect is still not understood. One hypothesis is that the presence of preformed amyloid deposits (nidus) in the heart and other organs of patients

before liver transplantation can lead to efficient recruitment of wild-type TTR in these deposits after orthotopic liver transplantation.30 In AD the most desirable approach, targeting the β- and γ-secretases to reduce amyloid β production, proves to be challenging. These enzymes have protein substrates other than amyloid precursor protein (e.g., neuregulin-1 for βsecretase, Notch1 for γ-secretase), and inhibiting the cleavage of these substrates has detrimental effects.31 Another therapeutic strategy is purposeful interference with the amyloid aggregation process. For example, eprodisate disodium (Figure 1, compound 1) is now in phase III clinical trial for the treatment of AA amyloidosis (ClinicalTrials.gov identifier NCT01215747). Compond 1 is a small sulfonated molecule which competes with sulfated glycosaminoglycans in binding to amyloidogenic proteins, a process that interferes with fibril formation and deposition. A phase IIa clinical trial for the treatment of type 2 diabetes has been completed as well (ClinicalTrials.gov identifier NCT00675857). However, the C



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Table 1. Recently Completed and Ongoing Clinical Trials for Nonantimicrobial Activities of TCs drug Doxycycline Minocycline, doxycycline Doxycycline Doxycycline Doxycycline Doxycycline

Doxycycline CMT-3b CMT-3b CMT-3b CMT-3b Doxycycline Minocycline Minocycline Minocycline Doxycycline Doxycycline

Minocycline

Minocycline Minocycline Minocycline Minocycline Minocycline Minocycline a

disease

effect

Inflammation in cystic fibrosis

MMP-9 inhibitory Anti-inflammatory Cerebral arteriovenous malformations and aneurysms MMPs inhibitory Lymphangioleiomyomatosis MMPs inhibitory Abdominal aortic aneurism MMP-9 inhibitory Post myocardial infarction remodeling MMPs inhibitory Anti-inflammatory Inflammation and insulin resistance in type II diabetes MMPs inhibitory and obesity Anti-inflammatory Persistent symptoms postneuroborreliosis Immunomodulatory Kasposi’s sarcoma MMPs inhibitory Recurrent brain tumors NSa Advanced solid tumors NSa Refractory metastatic cancer MMPs inhibitory Transthyretin amyloidosis NSa Huntington disease NSa Parkinson’s disease NSa Alzheimer’s disease NSa Systemic and localized amyloidosis Antiamyloidogenic Alzheimer’s disease (biomarkers evaluation) Antiamyloidogenic MMPs inhibitory Anti-inflammatory Amyotrophic lateral sclerosis Antioxidant Anti-inflammatory Antiapoptotic Acute hemorrhagic stroke Neuroprotective Reduction of chemoradiation effects NSa Schizophrenia Anti-inflammatory Antidepressant Atrial fibrillation after cardiac surgery Anti-inflammatory Retinal vein occlusion Anti-inflammatory Retinal detachment surgery Neuroprotective

current status

ClinicalTrials.gov identifier

Phase IV completed

NCT01323101

Phase Phase Phase Phase

I IV II completed III

NCT00243893 NCT00989742 NCT00538967 NCT00469261

Phase IV completed

NCT01375491

Completed Phase II completed Phase I/II completed Phase I completed Phase I completed Phase II Phase II/phase III Phase II completed Phase II Phase II Phase III

NCT01205464 NCT00020683 NCT00004147 NCT00003721 NCT00001683 NCT01171859 NCT00277355 NCT00063193 NCT01463384 NCT01677286 NCT00439166

Phase II completed

NCT00355576

Not yet recruiting Phase II Phase III

NCT01388127 NCT01636934 NCT01403662

Recruiting Phase I/II Phase II

NCT01422148 NCT01468831 NCT01297816

Not specified. b6-Deoxy-6-demethyl-4-dedimethylaminotetracycline.115

compound 2) was found to prevent the TTR protein misfolding as a result of its stabilization into the native tetrameric conformation. It inhibits formation of TTR amyloid fibrils and halts disease progression in patients with TTR amyloid polyneuropathy.35 In a pivotal phase II/III randomized, double-blind, placebo-controlled trial, neuropathy did not progress in 60% of patients who received 2 versus 38% of patients of the placebo group.36 Finally, generation of antibodies against amyloid fibrils and amyloid-like aggregates represents the third type of approach and is aimed at clearance of preformed amyloid deposits. This approach is based on active immunization with aggregated Aβ or passive administration of anti-Aβ antibodies. It was shown to be effective in mouse models of AD in which there were no adverse events.37 However, when AD patients were vaccinated, 6% developed severe meningoencephalitis.38 Clinical trials with passive immunotherapy are currently underway. Initial assessment of a limited number of patients with PET imaging using 11 C-labeled Pittsburgh compound B showed that the peripheral administration of a humanized anti-Aβ monoclonal antibody was associated with 25% reduction in cortical fibrillar Aβ load over 78 weeks compared with a placebo.31 However, no beneficial effect on memory and the mental functions as well as activities of daily living has been found in a recently completed clinical trial on mild to moderate AD.39

observed side effects such as myocardial infarction, myocardial ischemia, transient ischemic attack, and angina pectoris have prompted further review of this therapeutic approach.32 In a phase III trial on a large AD population the use of the Aβ ligand tramiprosate as an inhibitor of Aβ aggregation demonstrated no clear benefits.33 This result was in spite of the efficacy in preclinical animal studies, suggesting that great care is needed in attempting to extrapolate results from model organisms and computational models to the human situation. Overall, considerable effort in the design of Aβ targeting molecules has been useful, providing several classes of compounds with different modes of activity.33 The drugs selected for clinical trials, however, have produced unsatisfactory results or caused adverse side effects. In past decades efforts aimed at generating other small inhibitors of aggregation were also made. Thus, short β-sheet breaker peptides, iPrP13 and iAβ5, were developed for the treatment of prion and Alzheimer’s diseases, respectively.34 In animal models these peptides have been shown to inhibit fibrillogenesis and to induce disassembly of fibrillar amyloid deposits in vivo, resulting in a substantial delay in the disease onset. However, proteolytic degradation, generation of immunoreactive response, and the poor permeability of these peptides at the blood−brain barrier impede their application in medicine.34 Among other inhibitors, tafamidis (Figure 1, D



Perspective

attention to the structural features involved in their antiamyloidogenic activity. We discuss putative mechanisms underlying the beneficial effects of TCs on amyloidoses observed in vivo. Finally, we examine evidence of the possible synergic effect of pleiotropic nonantimicrobial activities of TCs on different pathological pathways of amyloidogenic disorders. The ultimate purpose of this review is to bring to the attention of scientists and clinicians the versatility of properties of TCs. We argue that the versatility of these properties should be taken into careful consideration in both preclinical and clinical studies.

Purified intravenous immunoglobulin (IVIg) obtained from the plasma of healthy humans contains naturally occurring antibodies against Aβ peptides. IVIg has been shown to ameliorate AD pathology in mildly affected patients, and some clinical trials have been conducted to evaluate the effect of IVIg in patients with mild to moderate AD (ClinicalTrials.gov identifiers NCT00818662, NCT01561053, NCT01736579) and mild cognitive impairment (ClinicalTrials.gov identifier NCT01300728). The nonfibrillar plasma glycoprotein serum amyloid P component (SAP) was found to be a component of amyloid deposits and might contribute to their persistence and resistance to proteolysis.1 The combination of the SAP-binding molecule (R-1-[6-[R-2-carboxypyrrolidin-1-yl]-6-oxohexanoyl]pyrrolidin-2-carboxylic acid) with anti-SAP antibodies was proposed as an antiamyloidogenic strategy. In this way the SAP-binding molecule depletes rapidly circulating SAP, while anti-SAP antibodies reach residual SAP in the amyloid deposits and promote their clearance.1,40 Phase I clinical trial to assess the pharmacokinetics of GSK2315698 (SAP depleter) and GSK2398852 (anti-SAP monoclonal antibody) and circulating SAP concentrations is now ongoing (ClinicalTrials.gov identifier NCT01777243). About 10 years ago tetracyclines (TCs), a well-known class of antibiotics (Figure 2, compounds 5−20), were found to inhibit the aggregation of prion protein fragments (PrP106− 126 and PrP82−146) and Aβ peptides. Furthermore, TCs destabilized scrapie prion protein (PrPSc) and Aβ aggregates and promoted their in vitro degradation by proteases.41,42 Later, the antiamyloidogenic effects of TCs on TTR, W7FW14F apomyoglobin, amylin, Huntingtin, α-synuclein, α2-macroglobulin, and poly-(A) binding protein nuclear 1 were studied and confirmed.43 Although the exact mechanism of antiamyloidogenic activity of TCs is still unknown, it is likely related to their ability to interfere with the formation of β-sheet structure. This is characteristic of all amyloidogenic proteins, a quality that makes them potentially effective against most types of amyloidoses.43,44 TCs have an advantage over other newly proposed antiamyloidogenic drugs because of their well characterized pharmacological and pharmacokinetic properties and relatively low toxicity. Some of them (for example, minocycline (Figure 2, compound 7)) efficiently cross the blood−brain barrier.45 This old class of antibiotics was nearly abandoned as a result of the development of resistant bacterial strains. Today, however, TCs are involved in more than 130 clinical trials unrelated to their antimicrobial activity.46 Among others, these include clinical trials focused on the beneficial effects of tetracycline derivatives (in particular doxycycline (6) and 7) on the outcomes of amyloidogenic disorders, including Alzheimer’s, Parkinson’s, and Huntington’s diseases and TTR amyloidosis (Table 1). An increase in the number of publications regarding successful application of TCs for the treatment of amyloidoses indicates a growing interest in this therapeutic approach. Moreover, the use of 6 as an orphan drug for the treatment of hereditary amyloid polyneuropathy caused by β2M was recently approved by the European Committee for Orphan Medicinal Products (http://www.emea.europa.eu/ docs/en_GB/document_library/Orphan_designation/2012/ 05/WC500127736.pdf). In this review, we first summarize data available in the literature regarding the antiamyloidogenic activity of TCs in cell-free, in vitro, and in vivo studies. We also give a brief insight into the chemical properties of these molecules, paying special

II. CHEMICAL STRUCTURE AND PHYSICOCHEMICAL PROPERTIES OF TETRACYCLINES Tetracyclines are a group of structurally related antibiotics discovered in the late 1940s (Figure 2). The first members of this family, chlortetracycline (14) and oxytetracycline (11), were isolated from Streptomyces aureofaciens and Streptomyces rimosus. They were later followed by the discovery of other natural TCs.47 Further chemical modifications of natural structures and synthesis of new compounds produced a number of novel TCs, including two of the more common semisynthetic clinical antibiotics 7 and 6.46 The basic chemical structure of TCs is represented by the partially saturated naphthacene carboxamide nucleus, composed of four linear fused six-membered carbocyclic rings A, B, C, and D (Figure 2, inset 2A). This structure is required for bioactivity in both prokaryotic and eukaryotic systems. Both natural TCs and many of their synthetic derivatives possess five asymmetric centers: C4, C4a, C5a, C6, and C12a. They have various functional groups attached to the periphery of the molecule.47,48 The lower and the upper peripheral regions are distinguished. Structure−activity relationships studies demonstrate that modifications along the lower periphery, rich in oxygen functional groups, cause a decrease in both antimicrobial and nonantimicrobial activities. Changes along the upper periphery, particularly in positions C7 and C9 of the D-ring, mostly result in increased activity against various biological targets. Modifications of the upper peripheral region influence basic physicochemical properties such as shape, electronic configuration, charge density and distribution, and polarity. Consequently these changes lead to changes in solubility in biological fluids and lipophilicity.48 The C4-dimethylamino group has proven to be necessary for antimicrobial activity. However, it is not involved in the biological effects on eukaryotic targets such as matrix metalloproteinases (MMPs).48 The presence of the phenol ring in the chemical structure of TCs makes them effective scavengers of reactive oxygen species (ROS), similar to α-tocopherol and other phenolic antioxidants. The reaction of TCs with free radicals results in formation of phenol-derived radicals. The latter radicals are relatively stable and unreactive due to their resonance and steric stabilization by phenol ring side groups.49 The scavenging potency strongly depends on the number and size of phenol ring substituents. In fact, in cell-free assays it was demonstrated that the presence of dimethylamino substituent at the C7 atom of 7 strongly improved its scavenging capacity of radicals compared to tetracycline (5).49 At the same time, under a variety of conditions, TCs can generate free radical species that damage surrounding macromolecules.48 Thus, in a biological environment TCs can auto-oxidize in the presence of divalent cations such as Co2+. This process generates electronically reactive species and E



Perspective

cytotoxins like O2•−, HO•, H2O2, and singlet oxygen 1O2. This reaction occurs in the dark and involves the electron-rich lower peripheral region.48 Tetracycline molecules, because of the flexible connection between ring A and ring B, may adopt two main conformations: an extended conformation and a folded conformation. It is generally assumed that in basic or nonaqueous solutions the molecule adopts an extended one. In this conformation, the B, C, and D rings approximately lie on the same plane and the C1, C2, C3, and amide carbons lie above this plane.50 In neutral to acidic solutions TCs adopt a folded conformation. This is derived from the extended conformation by the rotation around the C4a−C12a bond, causing the A-ring to be more folded toward the B-ring.50 The type of conformation is principally dependent on the protonation state of the molecule. In general, TCs possess some groups with acid−base properties (the C1− C3 tricarbonylmethane, the two C10−C12 ketophenolic hydroxyls, and the C4 dimethylammonium group) which can be subject to protonation−deprotonation equilibria. This results in formation of various protonation states, 3D conformations, and tautomers.51 The protonation scheme of TCs has been the subject of disagreements and intense discussion. Initially it was thought that TCs can only undergo three deprotonations. According to different research groups, the first deprotonation occurred at C3-OH, whereas the second and the third deprotonations affected 4-dimethylamino group or C10-OH or C12-OH.51 The semiempirical studies of TCs provide theoretical and experimental evidence that TCs can undergo four deprotonations, adopting 64 different tautomeric forms.51 The deprotonation of C10-OH (pK = 11.8) requires strongly basic conditions, far from the biological pH. But the ionizable groups of TCs may participate in the coordination of metal ions, particularly transition metal ions. The binding of metal ions to ionizable groups facilitates their deprotonation, giving rise to observed pK values shifted as much as several units toward lower values. It means that in the presence of complex multiligand and multimetal systems like biological fluids, the proton release of C10-OH cannot be excluded. In aqueous solution, the first deprotonation of the fully protonated species occurs at C3-OH. This results in formation of a neutral compound, represented by nonionized and zwitterionic tautomers (deprotonation constant pK1 ≈ 3.1− 3.5). The second and the third deprotonations affect the C12OH (pK2 ≈ 7.2−8.5) and C4-NMe2H+ (pK3 ≈ 9.0−10.8), respectively. Finally, the fourth acidic group that undergoes deprotonation is represented by the C10-OH (pK4 ≈ 11.8).51 In solution, TCs are an equilibrium of different tautomers, which can be shifted by varying the pH, the dielectric constant of the medium, and the metal ion presence. On the basis of the generally accepted attribution of the pKa values to specific functional groups, 5 in aqueous solution at neutral pH presents two different forms. The first is the zwitterionic form with a positive charge on the C4-NMe2 group and a negative charge on the C3-OH group. The second is the anionic form in which the C12-OH hydroxyl group is deprotonated (Figure 2).50 In addition, circular dichroism spectra of 11 demonstrate a solvent-dependent shift of the equilibrium between the zwitterionic and the nonionized forms. The zwitterionic form clearly predominates in aqueous solutions, while in organic solvents like ethanol, chloroform, and 1-octanol the nonionized form prevails.52 This nonionized, more lipophilic form of TCs easily crosses biological and model

membranes.52 The conformational flexibility of TCs makes it very difficult both to establish the structure−function relationship and to define their chemical behavior in a complex biological environment. On the other hand, the range of conformations and protonation states adopted by TCs in different chemical environments confers activity against many biological targets. The presence of different electron-donating groups in the chemical structures of TCs makes them powerful chelating agents. It is generally assumed that chelation sites include the deprotonated functional groups, primarily along the lower peripheral region.48,53 TCs bind with high affinity numerous polyvalent metallic cations, including Fe2+, Fe3+, Al3+, Ca2+, and Mg2+.48 In general, the affinity of TCs for different metals decreases in the following order: Fe3+ > Al3+ = Cu2+ > Co2+ = Fe2+ > Zn2+ > Mn2+ > Mg2+ > Ca2+ cations. For a given metal this ranking can vary, depending on pH, protonation state, the presence of other metal ions, and the presence of lateral substituents bound to the molecular tetracyclic skeleton.48,54 The results reported in the literature regarding the stoichiometries of complexes of TCs with metals, in particular with physiologically relevant Ca2+ and Mg2+ cations, are numerous and quite controversial. For both metal ions, stoichiometries of 1:1, 1:2, and 2:1 ions bound per 5 have been reported in aqueous solutions between pH 6.5 and pH 8.5.54 The results of the isothermal titration calorimetry studies suggest a distinct binding mode of TCs to Mg2+ as compared to Ca2+ cation.54 Moreover, it was shown that the molar ratios were distinct for the two ions, namely, 1:1 for the Ca2+/ tetracycline complex and 2:1 for the Mg2+/tetracycline.54 Metal chelation is relevant for a number of biological properties and activities of TCs. In blood TCs circulate primarily as complexes of calcium and magnesium.53 In the gastrointestinal tract TCs bind to polyvalent metal cations. They are normally present in biological fluids or ingested with food to form poorly absorbable complexes. This interaction, on one hand, may affect the biovailability of essential trace metal ions. On the other hand, it alters the biovailability of the antibiotics themselves. In fact, simultaneous ingestion of TCs with milk and other dairy products that contain metal cations such as calcium, magnesium, iron, and zinc may impair the absorption of the drug by 50−90%.55 TCs form a stable complex with calcium in any bone-forming tissue, tending to deposit in areas of calcification in bones and teeth. This accumulation may be associated with adverse effects such as permanent teeth pigmentation, especially in young individuals. Moreover, treatment of pregnant women with TCs may produce permanent brown discoloration of the deciduous and permanent teeth in their children because the drug passes the placental barrier. Importantly, this effect depends on the dose but not on the duration of theraphy.56 At the same time, the tendency of TCs to deposit in areas where new bones are mineralizing can be useful for studies of bone formation and turnover in vivo. 57 Thus, microscopic observation of fluorescence, derived from tetracycline−calcium orthophosphate complexes at sites of new bone formation, permits one to distinguish osteoid tissue from the mineralized bone. Moreover, observing the distribution of fluorescence derived from various TCs in different regions of the bone affords insight into the rate and extent of bone formation in vivo. This approach has found an application in clinical practice for characterization of dynamic changes in bone turnover in both normal and certain pathological conditions including hyper- and hypothyroidism,58 F



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postmenopausal osteoporosis, and myelodysplastic syndrome.59 Compound 5 labeling was also used for quick and easy intraoperative localization of the lesions to be resected in patients affected by osteoid osteoma.60 Chelated TCs can act as ionophores and transport bound calcium cations across biological membranes.48 This property has important biological implications because on one hand it can affect the release of calcium from storage organelles such as sarcoplastic reticulum, endoplastic reticulum, and mitochondria.48 On the other hand, once transported inside the cell by TCs, Ca2+ can act as a secondary messenger, affecting various biological pathways such as metabolic and secretory processes, cell division, receptor activation, and membrane transport. Metal chelation plays an important role in antimicrobial activity of TCs as well. Compound 5 chelates magnesium ion situated near the acceptor site for aminoacyl-tRNA of the 30S subunit of bacterial ribosomes. Such chelation interferes with the binding of aa-tRNA to ribosome by direct steric hindrance.61 As a result, protein biosynthesis is blocked, causing bacterial cell death. On the other hand, magnesium complexes of 5 play a crucial role in the mechanisms of bacterial resistance to the antibiotic. In complex with magnesium, the drug binds to the tetracycline repressor TetR and triggers its conformational changes, resulting in the expression of the gene tetA. The latter encodes for the membrane-bound tetracycline efflux pump, which exports the antibiotic out of the cell, thereby preventing bacterial death.48 In addition, inhibition of matrix metalloproteinase, a nonantimicrobial activity of TCs, is also strictly dependent on their metal chelation capacity. To summarize, it is clear that TCs possess pleiotropic biological properties. By means of a rational chemical modification of their structure, it is possible to generate compounds with low antimicrobial activity with enhanced therapeutic potential against nonbacterial biological targets. A substantial body of data available in the current literature provides a detailed description of structural and conformational behavior of TCs in various environments. However, to date, there is no consensus about what makes them efficient against such a variety of different targets. Elevated conformational versatility of TCs upon changes in surrounding conditions (pH, metal ion presence, dielectric constant of the medium, etc.) makes it very difficult to characterize them. It is likewise difficult to define the exact structure−activity relationships. At the same time, structural flexibility appears to be a key factor that confers activity against so many different targets. The high lipophilicity of some derivatives (i.e., compound 7) permits them to cross blood−brain barriers with relative ease. Another important feature is their chelation property, implicated in their interactions with various macromolecules of both bacterial and mammalian origin. Taken together, all these features provide TCs with great pharmacological potential for a variety of molecular targets and have given rise to numerous preclinical and clinical studies unrelated to their antimicrobial activity.

progression of amyloidosis and improved the clinical condition of five patients over the course of several months.62 Furthermore, in vitro studies have demonstrated that 3 bound strongly to five natural amyloid fibrils including amyloid A, LC, TTR, Aβ, and β2M but did not bind to native amyloid precursor.63 In vivo in experimental murine reactive amyloidosis 3 specifically accumulated in amyloid deposits. Taken together, these data suggested that the drug targeted certain structural features of amyloid fibrils, like cross-β-sheet quaternary structure.63 Quantitative binding studies on synthetic fibrils made of human recombinant insulin showed that 3 recognized with high affinity two unrelated binding sites (dissociation constants KD = 5.9 × 10−11 M and KD = 3.4 × 10−9 M, respectively) along the amyloid fibril.63 Compound 3 also bound to PrP amyloid in brain sections from scrapie-infected hamsters and patients with Creutzfeldt− Jakob disease (CJD). It reduced the infectivity of prion-infected brains, resulting in prolonged survival with experimental scrapie.64 In patients with FAP, 3 colocalized with amyloid deposits and in vitro disrupted TTR fibrils obtained from patients into an amorphous precipitate.65 Doxorubicin, which differs from 3 by the presence of the hydroxyl group at C-4′ of the amino sugar instead of iodine, showed much lower affinity for amyloid fibrils. However, the reason of the targeting of 3 to amyloid fibrils was not the presence of the iodine atom per se but the substantially increased hydrophobicity of 3 as compared to doxorubicin.63 All these data indicate the existence of a universal mechanism of action of 3 against amyloidosis and make it a potential antiamyloidogenic drug, feasible in all types of amyloidosis. However, because of the high toxicity (in particular, cardiotoxicity) of 3, attempts have been made to identify structurally similar compounds, possessing antifibrillogenic activity with a better safety/toxicological profile, already tested in clinical practice. The resemblance of the polycyclic conjugated structure of TCs with the aglycone moiety of 3 prompted an initial study to hinder the pathologic aggregation and the propagation of the prion protein.41,66 The advantages of TCs rely on their well characterized pharmacological and pharmacokinetic properties, relatively low toxicity, and the ability of some congeners to cross the blood−brain barrier.45 The first studies of antiamyloidogenic activity of TCs were carried out on fibrils generated by synthetic peptides corresponding to residues 82−146 and 106−126 of human PrP.41 These fragments have a high propensity to form insoluble fibrils with extensive β-sheet conformation. They are ultrastructurally similar to those found in patients with Gerstmann−Sträussler−Sheinken (GSS) and carry the protease resistant core of PrPSc.42,67,68 The interaction of 5 with PrP peptides was first established by fluorescence spectroscopic analysis, demonstrating a shift in the emission spectra of 5 in the presence of the peptides.41 This interaction resulted in inhibition of peptide aggregation into fibrils, causing an increase in protease sensitivity. It also abolished neurotoxicity and astroglial proliferation, induced by PrP 106−126 in vitro.41 Further studies showed that, like 5, 6 was able to revert the protease resistance of PrPSc protein from patients with CJD in a dose-dependent manner, reaching almost 80% at 1 mM.66 The pretreatment of scrapie−brain homogenates with 5 or 6 at 1 mM, followed by injection in Syrian hamsters, resulted in significant delay in disease onset and increased survival time.66 In addition, the ameliorations in pathological conditions in tetracycline-treated animals were associated with a delay in the accumulation of PrPSc in the brain. These findings were

III. ANTIAMYLOIDOGENIC ACTIVITY OF TETRACYCLINES The interest in antiamyloidogenic activity of TCs arises from the observation that a structural homologue of 5, the anthracycline anticancer drug 4′-iodo-4′deoxydoxorubicin (IDOX, Figure 1), produced clinical benefits in patients affected by immunoglobulin light-chain amyloidosis. Thus, a brief course (for about a month) of 3 (IDOX) inhibited G



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significant reduction of peptide toxicity as shown by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro.44 Compounds 5, 6, and 7 (50 μM) delayed the Aβ-induced paralysis in transgenic Caenorhabditis elegans strains, which express human Aβ 1−42 and represent a simplified invertebrate model of AD.72 This effect was dosedependent and similar for all three congeners. The beneficial effect on paralysis was associated with a strong decrease in the soluble oligomeric Aβ forms and reduced deposition of fibrils in muscle cells. Moreover, FTIR spectroscopy was used to characterize amyloid aggregates conformation at the molecular level in Caenorhabditis elegans and it was shown that TCs hinder protein aggregation. In particular, worms treatment with TCs significantly reduced the absorption in the amide I region of second derivative FTIR spectra (band at 1623 cm−1), which characterizes the intermolecular β-sheet structure of protein aggregates.72 The therapeutic role of 6 in combination with another antibiotic, rifampin (Figure 1, compound 4), that was shown to inhibit Aβ aggregation and in vitro neurotoxicity has been assessed in patients with AD in a randomized, placebocontrolled, multicenter clinical trial.73,74 It has been shown that a 3-month course therapy of 6 (200 mg) and 4 (300 mg) significantly reduced cognitive worsening at 6 months of follow-up in patients with mild to moderate AD.73 There was also less dysfunctional behavior at 3 months. This decrease was not, however, observed at 6 and 12 months in the treatment group with respect to the placebo group.73 Since Chlamydia pneumonia infection is thought to be implicated in AD, the combined treatment with compounds 4 and 6 on Chlamydia pneumonia IgA or IgG antibody titers in patients was performed. However, the laboratory results did not show any significant reduction of Chlamydia pneumonia IgA or IgG antibody titers between treatment groups at 6-month follow-up. Considering previously reported antiamyloidogenic activity of both 6 and 4, authors suggested that the effect of the drugs on cerebral amyloid deposition could explain the clinical findings.73 However, the subsequent multicenter, blinded, randomized, 2 × 2 factorial controlled trial, conducted by the same group and involving 406 patients, demonstrated that 12 months of treatment with 6 (100 mg twice a day) or 4 (300 mg/day) had no beneficial effects on cognition or function in AD.74 Currently, there are two ongoing clinical trials studying the effect of TCs in patients with AD (Table 1). These results attracted the interest of the research community and gave rise to various in vitro and model organism studies using amyloidogenic proteins including but not limited to human amylin, huntingtin, W7FW14F, apomyoglobin, poly-(A) binding protein nuclear 1, α-synuclein, β2M, and LC. Thus, Aitken et al. found that 5 decreased fibril formation by the pancreatic hormone amylin, representing the pancreatic islet amyloid in advanced type II diabetes mellitus.75 In vitro studies showed that the presence of 20-fold molar excess of 5 caused a gradual reduction in ThT binding to amylin with a half-life of 3.4 h. This indicates the drug’s capacity to hinder amylin assembly. Transmission electron microscopy experiments have complemented these data, showing that incubation of synthetic human amylin with 20-fold molar excess of 5 for approximately 20 h resulted in the formation of small globular structures and short fibril fragments. In the absence of the drug, however, longer and more dense characteristic amylin fibrils were observed.75 Successive in vivo studies were carried out on transgenic hA/hIAPP mice that spontaneously develop

revealed by immunohistochemistry and Western blot analysis in the post-mortem brains. These data were also confirmed by magnetic resonance imaging (MRI) studies in animal models of prion disease, demonstrating that the extent of PrP Sc accumulation in the brain was remarkably more abundant in the positive controls than in tetracycline-treated hamsters. The observed differences between the two groups were paralleled by a difference in the severity of spongiform changes and astrogliosis in the cerebral cortex and subcortical gray structures.66 Compound 5 was found to bind not only to PrP aggregates but also to oligomeric−monomeric forms of PrP106−126,41 as revealed by fluorescence microscopy and nuclear magnetic resonance (NMR) spectroscopy. In particular, NMR data provided evidence for through-space interactions between the aromatic protons of 5 and hydrophobic peptide domains. This included the side chains of Ala117−119, Val121−122, and Leu125, all involved in the formation of βsheet secondary structure.41 At the same time, fluorescence experiments, together with docking calculations and molecular dynamics simulations, suggested that antibiotics can also interact with the solvent-exposed C-terminal helix 2 of human PrP. This fragment of PrP is a potential nucleation site of the conversion from the cellular to the scrapie form of PrP. Furthermore, it can adopt both α- and β-structure without a definitive preference.69 These data suggest that TCs not only inhibit protein aggregation and fibrillization through interaction with β-sheet forming domains but also stabilize the fluctuating conformation of the C-terminal part of helix 2 and prevent conformational transition of PrP to the pathological isoform.69 In addition, circular dichroism studies showed that 6 and 5 prevented formation of β-sheet structures by human recombinant PrP91−230, a starting event of the conversion of amyloidogenic proteins into fibrils.70 Finally, the anti-prion activity of TCs has been studied in a small group of patients affected by CJD. These patients received compassionate daily treatment with 6 at 100 mg/kg.71 Retrospective analysis showed significantly longer survival in these patients, which in turn gave rise to the ongoing phase II, multicenter, randomized, double-blind, placebo-controlled efficacy study of 6 in CJD patients funded by the Italian Drug Agency.71 Antiamyloidogenic activity of both 5 and 6 has also been tested on synthetic peptide amyloid β 1−42 (Aβ 1−42), representing the major component of amyloid plaques in AD.42 Electron microscopy studies, together with quantitative ThT assay and trypsin digestion studies, have shown that equimolar concentrations of TCs not only inhibited amyloid fibrillogenesis but also disrupted preformed amyloid fibrils.42 Two biophysical methods that permit following of the interaction between ligand and proteins, NMR and Fourier transform infrared spectroscopy (FTIR), have demonstrated that 5 binds to the Aβ peptide in a nonspecific manner. This was evidenced by the absence of a well-defined binding site on the peptide.44 Atomic force microscopy (AFM) and dynamic light scattering analysis showed that the supermolecular complexes between the drug and Aβ formed immediately after their co-dissolution. The resulting aggregates formed by peptide with 5 were larger than those detected in the absence of the drug (peptide alone 1.56 nm vs peptide−5 2.94 nm) and increased slowly with time to reach a maximum after 24 h. In fact, after 120 h of incubation with 5, larger clusters of about 17 nm were observed. These colloidal particles were very stable and did not show any laterstage evolution. They sequestered oligomers and prevented further progression of the amyloid fibril growth, resulting in H



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(Clinicaltrials.gov identifier NCT01171859) to evaluate the efficacy, tolerability, safety, and pharmacokinetics of 6 (100 mg twice a day) and TUDCA (250 mg three times a day) administrated continuously in patients with TTR-related amyloidosis.80 The results of the 12-month follow-up show that coadministration of 6 (100 mg twice a day) and TUDCA (250 mg three times a day) stabilized the disease for at least 1 year in the majority of patients treated to date. No clinical progression of cardiac involvement was observed. The neuropathy, as well, remained substantially stable for over 1 year. The treatment was well tolerated and feasible for aged patients with advanced diseases.80 Human α-synuclein is a highly conserved presynaptic protein. When aggregated, it represents the major fibrillar component of cytosolic filamentous inclusions in α-synucleopathies, including Parkinson’s disease (PD).81 Thioflavin S fluorescence and electron microscopy studies have shown that 5 dose-dependently inhibited fibrillogenesis of α-synuclein and destabilized preformed fibrils in vitro.81 Compound 7 was shown to be protective in animal models of PD, induced by 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine, providing dopamine depletion.82 It was also tested in an 18-month doubleblind futility clinical trial for its potential to alter the short-term course of PD to a predetermined threshold.83 This approach permits minimizing the number of subjects required for the study and provides indications of whether or not to proceed with further large-scale trials. The results of the 12-month evaluation of the clinical progression of PD suggest that 7 should be considered for definitive phase III trials to determine if it alters the long-term progression of the disease.83 Beneficial effects of 7 in a mouse model of Huntington’s disease (HD), characterized by formation of Huntingtin aggregates in autopsy brains, were first reported in 2000 by Chen et al.84 Thus, in R6/2 transgenic mice, expressing exon-1 of human huntingtin, daily treatment with 5 mg/kg of 7 resulted in improvement of rotarod performance and extended survival rates by 14%. These ameliorations were strongly associated with minocycline-induced inhibition of caspase-1 and caspase-3 up-regulation and reduced activity of inducible nitric oxide synthase (iNOS).84 Later, Smith et al. investigated the ability of TCs to inhibit Huntingtin aggregation both in vitro in a hippocampal slice culture and in vivo using the same mouse model. This was done in order to estimate the contribution of antiamyloidogenic activity of TCs in neuroprotection.85 It was found that 5, 6, and 7 drastically reduced aggregation of huntingtin at 30 μM in organotypic slice culture model. They were also well tolerated and reduced hyperglycemia in aged R6/2 mice by an unknown mechanism. However, in contrast to previous studies, the drugs neither reduced aggregate load in R6/2 brains nor produced any improvement in the behavioral phenotype of mice.85 Another mouse model of HD (strain N171-82Q) also reported no improvements in survival, weight loss, or motor function after treatment with 7.86 Finally, to assess the safety and futility of 7 at 200 mg/day in HD, an 18-month randomized, double-blind study using a futility design was conducted by the Huntington Study Group.87 This study demonstrated that while not entirely futile, treatment with 7 provided minimum clinically relevant benefit. In conclusion, the results suggested that it would not be advisable to continue with the larger trial.87 Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant progressive disease. It is associated with accumulation of fibrils, formed by the mutant poly-(A) binding

diabetes and produce human amylin (hA)/islet amyloid poypeptide (hIAPP), thereby forming amyloid deposits in pancreatic islets.75 In this model, low doses of 5 (0.03 mg/mL drinking water) for 60 days partially suppressed the progression of diabetes. However, it did not influence the disease onset. Administration of 5 at a higher dosage (0.5 mg/mL drinking water) for 60 days significantly delayed both disease progression and onset. In particular, 5 dose-dependently ameliorated both hyperglycemia and polydipsia, resulting in an increase in median survival of 254% compared with control animals.75 The authors suggest that the observed ameliorations were likely linked to the interaction of 5 with soluble nascent prefibrillar aggregates of hA/hIAPP rather than to any putative extrapancreatic effects.75 Similarly, 5-fold molar excess of 5 inhibited fibrillogenesis of the amyloid forming protein W7FW14F apomyoglobin.76 AFM, ThT fluorescence, and dynamic light scattering studies have demonstrated that incubation of the protein for 7 days in the presence of 5 inhibited the formation of fibrils from early aggregates. In fact, in the presence of 5 only globular oligomeric structures were observed, while control samples presented mostly mature fibrils. It is worth noting that, once formed, mature fibrils of W7FW14F apomyoglobin could not be disrupted by 5 even at high drug to protein molar ratios (up to 25:1) or long incubation time (up to 15 days). This was at variance with previously reported results for Aβ 1−42, PrPSc, and TTR.42,66,77 According to the authors only few segments of W7FW14F apomyoglobin are involved in the formation crossβ-sheet structure. Therefore, the core of the fibril might have low accessibility for the noncovalent interactions with 5, thus preventing fibril disaggregation by the drug.76 It was also found that the persistent globular aggregates of W7FW14F apomyoglobin forming in the presence of 5 were able to interact with cell membranes and cause cytotoxicity. In this regard, authors suggest the careful usage of 5 as fibril inhibitor.76 Compounds 5, 6, 7 and rolitetracycline (17) are all able to disrupt fibrils of TTR Leu55Pro protein, associated with one of the most aggressive forms of FAP and composing of extracellular amyloid deposits.77 Thus, after 17 days of incubation at 37 °C with the drugs, the samples clearly showed an abundance of small round particles. In these conditions 6 was revealed as the most effective of the tested derivatives, as no fibrils were visible after incubation while in the cases of 5, 7, and 17 very few fibrils were present, similar to those found in control samples.77 Fibril disrupting activity of 6 has been confirmed in vivo in transgenic mice caring the Val30Met mutant of TTR.78 Thus, animals treated with 40 mg kg−1 day−1 6 in drinking water for 3 months did not present mature TTR fibrils in deposits, as established by the lack of green birefringence in tissues stained with Congo red. At the same time, levels of nonfibrillar TTR load in tissues of treated animals were comparable to those detected in the control group. This suggests that while 6 was very efficient at disrupting already formed fibrils, it could not inhibit fibril formation until they reach a certain length.78 However, combined administration of 6 and an antioxidant compound, tauroursodeoxycholic acid (TUDCA), in transgenic mice produced a synergic beneficial effect on both TTR amyloid deposition and all biomarkers associated with FAP.79 This stands in contrast to single individual drug administration which did not affect biomarkers.79 On the basis of these observations, Obici et al. designed a phase II, open-label study I



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the doxycycline-induced evolution of fibril morphology imaged by AFM. In fact, exposure to 6 gave rise to fibril disaggregation into smaller oligomeric species. These accumulations peaked after 3 days of incubation. At longer incubation times, these oligomers reassembled into more complex structures.91 Curiously, among 13 tetracycline congeners tested, only 7 derivatives were able to inhibit β2M fibril growth, as established by ThT assay. According to these data, antifibrillogenic activity of these derivatives decreases in the following order: 6 ∼ 4epioxytetracycline (13) > 11 ∼ methacycline (9) > meclocycline (8) > 17 ∼ anhydrochlortetracycline (16). Compounds 5, 7, 14, demeclocycline (15), 4-epichlortetracycline (18), and 4-epianhydrotetracycline (12), on the other hand, were not active.91 Interestingly, according to different studies, antiamyloidogenic activity of TCs varies significantly. By estimation of the ability of 15 TCs to reduce the resistance of preformed PrP106−126 fibrils to proteinase K, 11 and 17 were identified as the most active derivatives. Compounds 5, 6, anhydrotetracycline (10), 13, and 15 were less potent, while 7 and some other analogues were not active.92 Another study, based on Aβ-fibril specific immunoassay, showed that only one (17) out of eight compounds was able to inhibit formation of Aβ 1−40 fibrils.93 The immunoassay, based on estimation of the reduction of anti-Aβ antibodies binding to Aβ 1−42 fibrils in the presence of TCs, demonstrated that 11 and 14 bind more efficiently to Aβ fibrils compared to 5. Compounds 6 and 17 did not affect the binding.94 Electron microscopy images, obtained from TTR fibrils after 17 days of incubation with four TCs, revealed that 6 was the most effective compound. Compound 7 also demonstrated significant disrupter activity, apparently higher than both 5 and 17. The last one was the least potent fibril disrupter.77 However, in these studies different experimental conditions (such as initial state of aggregation, protein concentration, buffer, time of incubation), and approaches for detection of the effect were used. Therefore, it is difficult to explain the discordant results and to define unequivocally the structural features responsible for the antiamyloidogenic activity. On one hand, inhibition of amyloidogenesis could be a function of a specific protein structure, as was suggested by Giorgetti et al.91 On the other hand, false positive/negative results can be derived from the choice of an inappropriate method of screening. Thus, ThT binding assay is one of the most common approaches for studying amyloid aggregation. Recent NMR data, however, showed that 5 competes with ThT for the binding to protein,44 and hence, the use of another screening approach is preferable. For example, X-ray diffraction,5 ELISA-based assay,94 and MALDI MS based method95 for screening the efficiency of potential inhibitors of amyloid aggregation have all been utilized. At present, there are no clear data regarding the relationship between the structures of TCs and their antiamyloidogenic activity. The results of molecular mechanics investigations showed that despite the flexibility between the extended and folded conformations of TCs, there was no relevance to the observed activity. In fact, in solution TCs acquire different conformations and tautomeric forms that essentially depend on the solvent, pH, and metal ions such as Ca2+, Mg2+, Cu2+, Co2+, and Ni2+. This confers to the hydronapthacene moiety an extraordinary conformational flexibility, and as a consequence, QSAR studies could not identify any geometrical pharmacophore.50 Furthermore, three-dimensional quantitative structure−activity relationship (3D-QSAR) investigation has

protein nuclear 1 (PABPN1) within the nuclei of skeletal muscle cells. In a mouse model of OPMD, 6 not only delayed disease onset and attenuated disease phenotype but also decreased formation of PABPN1 aggregates in muscles.88 Since previous studies have demonstrated the positive correlation between the decrease in aggregation of PABPN1 and reduced cytotoxicity, authors suggested that the antiamyloidogenic properties of 6 likely contributed to its beneficial effects. Additionally, proapoptotic processes, such as Bax expression, cytochrome c release, and caspase-3 activation, were attenuated in response to the treatment with 6. This indicates that together with antiamyloidogenic properties, antiapoptotic activity of the drug could also play an important role in protection against OMPD.88 The most commonly diagnosed form of systemic amyloidosis results from the aggregation and deposition of LC, produced by clonal plasma cells in the bone marrow.89 About 26% of transgenic mice CMV-λ6, expressing human λ LC protein, developed amyloid deposits in the stomach within 6 months. Treatment with 0.5 mg/mL 6 in drinking water, starting from 3 to 6 months and lasting at least 7 months, resulted in reduced amyloid deposits. Thus, only 23% of the treated mice had amyloid deposits in the stomach, compared to 69% in the untreated control group.89 Electron microscopy studies have demonstrated that 6 not only inhibited fibrillogenesis of the recombinant LC protein in vitro but also disaggregated mature fibrils from patients ex vivo. Thus, after 5 days of incubation of the recombinant LC protein in water, EM images revealed the presence of mature fibrils. On the other hand, after coincubation with 6, fibrils were disorganized, broken, and left with frayed ends. Numerous large aggregates were also detected after incubation of tissue-extracted fibrils with 6, confirming a direct interaction between the drug and the fibrils.89 Abnormal aggregation and accumulation of a mutant αBcrystallin protein in myocytes is a key feature of desmin-related cardiomyopathy.90 Transgenic mice, producing a well characterized mutant of human αB-crystallin, CryABR120G, were used to test its therapeutic effect in cardiac proteinopathies. It was found that the administration of 6 mg/mL 6 with drinking water, starting at 8−16 weeks of age, significantly delayed the premature death of treated animals compared to control and attenuated cardiac hypertrophy. These ameliorations were associated with a decrease in accumulation of CryABR120G aggregates in the hearts of treated mice. Similarly, a dosedependent inhibition of aberrant protein aggregation by 6 was observed in culture cells infected with recombinant adenovirus expressing CryABR120G protein.90 Amyloid deposits formed in patients subjected to long-term hemodialysis are mostly composed of full length β2M and its N-terminal truncated species ΔN6β2m.91 Compound 5 and its derivatives were able to inhibit fibrillogenesis and, upon prolonged exposure, disaggregated β2M fibrils. This was shown by significantly reduced ThT fluorescence and complete loss of fibrillar structure observed by electron microscopy. Moreover, 6 at equimolar concentrations to β2M (100 μM) fully abrogated the toxicity of soluble β2M oligomers in SHSY5Y neuroblastoma cell line. The effect on the cytotoxicity of the mature β2M fibrils was more complex. In particular, the exposure of originally nontoxic fibrils to 6 elicited a transient dose-dependent cytotoxic effect in the range 100−300 μM. At 300 μM 6 this effect reached maximum levels after 3 days of incubation and disappeared after 7 days of incubation.91 Cytotoxicity was directly related to J



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involved in a variety of physiological and pathological processes, including wound healing, tissue repair and remodeling, embryogenesis, inflammation, vascular and autoimmune disorders, cancer progression, and metastasis.46,101−103 Elevated expression of MMPs is also strongly associated with amyloidogenic diseases. Thus, significantly increased levels of serum MMP-3 have been detected in hemodialysis patients with dialysis-related amyloidosis.104 Moreover, serum MMP-3 levels correlated with serum levels of β2-microglobulin, suggesting a direct stimulation of MMP-3 by β2M.104 In FAP the elevated expression of MMP-9, colocalized with TTR deposits, was detected by immunostaining salivary gland biopsies from patients affected by FAP. In control biopsies MMP-9 staining was absent.105 The overexpression of MMP-9 was observed only at a later stage of disease, when the extracelluar deposits of amyloid fibrils were already present.105 In patients with cardiac amyloidosis due to LC protein, the increased levels of serum MMP-2 and MMP-9 correlated with diastolic dysfunction.106 Aberrant and chronic overexpression of MMPs is also associated with the pathophysiology of neuroinflammatory processes accompanying most central nervous system disease. In particular, up-regulation of MPPs (MMP-9, MMP-3, and MMP-2) was detected in the plaqueaffected brains and blood plasma of AD patients.107,108 The overexpression of MMPs in AD is likely a part of an inflammatory response and can be induced endogeneously by amyloid.107 In vitro studies have shown that both MMP-2 and MMP-9 could degrade Aβ. It is thus believed that both MMP-2 and MMP-9 play a beneficial role in the extracellular Aβ catabolism and clearance in vivo. In a mouse model of AD MMP-9 is capable of degrading compact amyloid plaques in brain sections.109 Excessive activation of MMPs, however, could cause damage to the blood−brain barrier by attacking the extracellular matrix, basal lamina, and tight junctions in endothelial cells, thus promoting neurodegeneration.107,108 In addition, MMPs may act as signal molecules in both neuronal apoptotic processes and inflammation.110 For example, MMP-3 participates in neuronal apoptotic signaling by activation of caspase-3 followed by DNA fragmentation.110 In neuroinflammation MMP-3 activates microglia, leading to the release of proinflammatory cytokines such as IL-1β and tumor necrosis factor α (TNF-α).108 These molecules in turn contribute to the death and removal of damaged neurons under physiological conditions. In contrast, under pathological conditions, the overexpression of MMP-3 likely causes the death of nearby and undamaged neurons. An increase in MMP-3 levels has been observed in various experimental models of PD in the plasma, cerebrospinal fluid, and around senile plaques of AD patients. It also appears elevated in blood mononuclear cells of multiple sclerosis patients.108 Taking these facts into account, down-regulation of MMPs is expected to produce beneficial effects in a number of pathological conditions. A considerable effort has been made to develop safe and effective synthetic compounds targeting MMPs. Nearly 60 MMP inhibitors, including peptidomimetics, nonpeptidomimetics, and various natural compounds, have been developed and tested in clinical trials over the past several years. With the noteworthy exception of 6, however, all MMPs inhibitors were abandoned at early stages of clinical trials because of low water solubility, lack of efficacy, and severe side effects. This failure is mainly due to the broad spectrum of action of MMPs inhibitors and complex biology of the enzymes.101

revealed that hydroxyl groups at positions 5 and 6 are important factors in determining antifibrillogenic activity. This is due to their ability to provide donor sites for putative H-bond interactions with PrP peptides.92 These results are in agreement with data reported by Giorgetti et al. that suggest the relevance of the C5 oxidryl group for the interaction of TCs with β2M.91 A second relevant factor, according to 3D-QSAR calculations, is that the presence of electron-donor substituents on the D-ring enhances the π-electronic configuration of the aromatic ring and favors π-stacking interactions with electron deficient peptide sites. The correct spatial orientation of the positively charged C4-NMe2 group contributes to activity, ensuring electrostatic interactions with negatively charged sites. Alkylamine substituent at the amidic group in position 2, as in the case of 17, was indicated as another factor enhancing antiamyloidogenic activity, while the keto−enolic switch between positions 11 and 12 decreases this activity.92 To conclude, numerous studies have demonstrated the antiamyloidogenic activity of TCs both in vitro and in vivo. In particular, it has been established that (i) TCs bind to the βsheet forming domain of all amyloidogenic proteins, independent of their primary structure, by nonspecific interactions,41,44,91 (ii) inhibition of fibrillogenesis is a result of the immediate interaction of amyloidogenic protein with the drug, resulting in formation of stable globular oligomeric species and preventing further fibril growth,44 (iii) TCs also affect mature fibrils, resulting in their complete or partial disaggregation,42,75−77,81,85,89−91 (iv) treatment with TCs reduces amyloid-induced toxicity in cell culture,41,44,66,72,79,84 (v) treatment with TCs is often associated with reduced amyloid load in tissues and provides both ameliorations in pathological symptoms and prolonged survival in animal models of amyloidoses,17,82,84,88−90 (vi) several preliminary and ongoing clinical trials demonstrate improvements in clinical outcomes in patients affected by different types of amyloidoses in response to treatment with TCs.73,80,83,87

IV. OTHER NONANTIMICROBIAL ACTIVITIES OF TETRACYCLINES AND THEIR POSSIBLE IMPLICATION IN THE TREATMENT OF AMYLOIDOSES It is well-known that extracellular deposition of protein fibrillar aggregates is the main hallmark of amyloidogenic disorders. Since a variety of studies have demonstrated the ability of TCs to disrupt and/or inhibit formation of amyloid fibrils, this mechanism was initially thought to be responsible for the beneficial effects of TCs in amyloidoses.17,66,88,90 At the same time, these diseases are also associated with other common pathological features such as inflammation,10,80,96 ROS generation causing oxidative stress,10,97 dysregulation of metal homeostasis,98 mitochondrial dysfunction,99 and apoptosis.100 TCs have been recognized as therapeutic agents affecting a variety of pathological targets.46,100 These include matrix metalloproteinases, apoptotic and inflammatory pathways, and oxidative stress. Therefore, in order to gain a better understanding of the mechanisms underlying the beneficial effects of TCs in amyloidoses, it is necessary to first summarize the main features of nonantimicrobial activities of TCs.46 1. Inhibition of Matrix Metalloproteinases. Inhibition of MMPs is probably the best characterized nonantimicrobial property of TCs. MMPs are a family of zinc-dependent extracellular matrix remodeling endopeptidases. They are K



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In 1983 Golub et al. first reported their finding that TCs are capable of inhibiting collagenase-mediated breakdown of connective tissue in periodontitis that is associated with type I diabetes.111 Later, Greenwald et al. found that 7 could reduce excessive collagenase activity in diseased joints of patients with rheumatoid arthritis.112 While the precise mechanism underlying the inhibitory effect of TCs on MMPs is not yet clear, it is believed that TCs act by direct inhibition of the enzymes and by down-regulation of their expression.46 Direct inhibition of the MMPs activity is likely mediated by TC chelation of metal ions associated with the enzyme. This process can, in fact, be reversed by the addition of micromolar concentrations of zinc or millimolar concentrations of calcium ions.113 Moreover, it was demonstrated that a chemically modified derivative of 5 (tetracycline pyrazole, CMT-5,115 compound 19) incapable of metal chelation did not inhibit MMPs.114 It was initially thought that 5 binds catalytic Zn2+. Further structural studies, however, indicated that the mechanism of inhibitory effect involves drug interaction with the structural zinc and/or calcium atoms within the protein rather than with the catalytic zinc ions.113,115,116 In fact, Garcia et al. demonstrated that 6 binds matrilysin (MMP-7) proximal to the structural metal center of the protein, containing both zinc and calcium ions, and destabilizes the tertiary structure of the enzyme.116 Moreover, studies of the effect of 6 on MMP-8 activity and susceptibility to proteolytic digestion indicate that the destabilization of the enzyme is due to binding of TCs to calcium rather than to zinc ion.117 In fact, the inhibitory effect of 6 on MMP-8 was duplicated by calcium and zinc chelator EDTA. This effect was not duplicated, however, by 1,10phenanthroline, a selective chelator of zinc.117 Compound 6 is now the only inhibitor of MMPs approved for systemic use by the U.S. Food and Drug Administration and regulatory agencies in Europe and Canada.118 The beneficial effects of TCs as MMPs inhibitors are now being studied in a number of clinical trials (Table 1). Various clinical studies evaluating beneficial effects of TCs in rheumatoid arthritis demonstrated mild to moderate beneficial effects.119 In a very recent paper, however, Greenwald affirms that the road forward in rheumatoid arthritis treatment no longer involves 6 or 7. He pointed instead to other, more powerful drugs. His conclusions do not exclude the possibility that a more effective chemically modified nonantimicrobial chemically modified derivative of tetracycline (CMT) may prove to be useful.119 2. Scavenging of Reactive Oxygen Species. Oxidative stress is associated with increased formation of ROS, resulting in DNA damage, lipid peroxidation, protein modification, and other effects. It is also a typical feature of numerous diseases including cancer, cardiovascular disease, diabetes, atherosclerosis, neurological disorders (AD, PD etc.), and chronic inflammation.120 Reactive oxygen species, including superoxide (O2), hydrogen peroxide (H2O2), peroxynitrite (ONOO−), hydroxyl radical (OH•), and nitric oxide (NO), are molecules with unpaired electrons in their outer orbit. This makes them very unstable and highly reactive, disposed to initiate chain reactions with surrounding biomolecules. For example, peroxynitrite and its decomposition products can modify the structure and function of proteins by nitration of tyrosine residues or methionine sulfoxidation.121 At lower concentrations NO acts as a neurotransmitter and signaling molecule.122 It is estimated that generally 5% of oxygen taken up by a tissue is transformed into ROS. In order to overcome

the potential toxicity of ROS under physiological conditions, they are neutralized by endogenous enzymatic free radical scavengers such as superoxide dismutase, glutathione peroxidase, and catalase.120 Under pathological conditions, an excessive production of ROS overwhelms the defense capacity of endogenous antioxidants and may produce injurious radicalmediated reactions causing cell apoptosis. In order to reduce the oxidative stress and ameliorate the pathological conditions of some ROS-related diseases, antioxidant therapeutics have been proposed. These include small ROS scavengers, inhibitors of ROS generating enzymes, and antioxidative enzymes.120 Induction of inducible nitric oxide synthase (iNOS), responsible for excessive nitric oxide and peroxynitrite production, has been observed in animal models of AD and HD, as well as in humans affected by FAP, AD, and HD.84,122,123 Some data indicate that mitochondrial dysfunction likely underlies oxidative damage in AD brains.97,124 Those neurons immunoreactive-positive for oxidative damage (8-hydroxyguanosine) also showed an increased cytochrome oxidase in the brains of AD patients, suggesting that mitochondrial dysfunction might lead to oxidative damage in AD. The antioxidant potential of TCs was first reported about 25 years ago.125 At that time it was shown that 5, 6, and 7 inhibited production of O2−, H2O2, and OH• by zymosan-stimulated leukocytes. Compound 7 was the only derivative able to scavenge free radicals directly in the cell-free xanthine−xanthine oxidase assay.125 Later, Akamatsu et al. demonstrated that 6 was also an efficient antioxidant in both cell-based and cell-free systems.126 In vitro studies have shown that TCs can protect biological macromolecules against damage induced by reactive nitrogen species, especially peroxynitrite. Moreover, recent studies showed that 7 acts as a direct and highly selective scavenger of peroxynitrite.121 The same study demonstrated that 5, however, did not. According to the authors, peroxynitrite might be a direct target of 7 in vivo. They also suggested that peroxynitrite neutralization by 7 represents the mechanism that protects biological targets from oxidative damage.121 At the same time, the antioxidant potential of 7 against other free radicals such as 6-hydroxydopamine, NO, and glutamate has also been observed in vitro.49 In vitro in a rat model of cardiac ischemia−reperfusion, 7 produced a cardioprotective effect in part due to the reduction of oxidative stress.127 Antioxidant properties of 6 have also been reported. Thus, in diabetic animal models and diabetic humans the presence of high glucose levels induced a significant increase in ROS production and impaired antioxidant defenses, causing severe cardiovascular dysfunction. Treatment of diabetic rats with 6 resulted in significant reduction in oxidative stress markers in plasma. It also preserved endothelial vascular functioning directly affecting endothelium, smooth muscle, or both.128 Moreover, 6 was shown to alleviate doxorubicinimposed oxidative stress on the heart tissue in mice by inhibiting ROS generation and stimulating endogenous antioxidant enzymes (superoxide dismutase and glutathione peroxidase).129 The mechanism of the antioxidant activity of TCs remains elusive. On one hand, the presence of a multiple-substituted phenol ring in the chemical structure of TCs makes them direct radical scavengers, similar to vitamin E.49,121,125 Reaction of the phenol ring with free radicals results in the formation of a relatively unreactive phenol-derived free radical.48 Stability of these radicals depends on the number and size of phenol ring substituents as well as the extent of resonance stabilization. In L



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arteries, 6 inhibited T cell activation and TNF-α production in peripheral immune cells.135 Enzymatic activity of MMP-9 induced by TNF-α also decreased after treatment with 6, reducing the elastin breakdown in coronary vessels and improving coronary outcome.135 Aortic aneurysm is another pathology strongly associated with inflammation and increased MMPs expression. Animal studies of 6 for abdominal aortic aneurysm treatment provided significant evidence of a beneficial effect. In mice with induced aneurysm a treatment with 6 produced a dose-dependent attenuation in the progression of aneurysmal dilatation.136 In humans there was a significant attenuation of vascular inflammation. This consisted of a reduction of neutrophil and cytotoxic T-cell content in the aortic wall, selective suppression of inflammatory cytokines (IL-6 and IL-8), transcriptional factors (AP-1, C/EBP, STAT3), and a reduction of neutrophilderived proteases (MMP-9). However, there was no amelioration in patients’ outcome.137 The conflicting evidence produced by human and animal studies regarding the beneficial effects of 6 on aneurysm progression is likely due to the poor quality of current clinical studies. It is expected that further clinical trials, involving large subject numbers with longer 6 exposure and prolonged follow-up, could help clarify this matter. At the same time, TCs were shown to produce beneficial effects in a number of inflammation-related pathologies. In a patient affected by pulmonary, ocular, and muscular sarcoidosis, 4-month treatment with 7 (200 mg/day) reduced both the muscle symptoms and the levels of IL-12 and interferoninducible protein-10 (IP-10), generated by granuloma macrophages at the inflamed sites and the bloodstream.138 Moreover, it was noted that during the treatment with 7 the levels of circulating cytokines and chemokines associated with T helper type 1 response decreased. Levels of Th2-associated chemokines remained elevated. The disease rapidly relapsed after discontinuation of the 7 administration. Subsequent 7 readministration resulted in prompt improvement of symptoms again, indicating the immunomodulatory mechanism of action.138 Similarly, some patients with persistent postneuroborreliosis symptoms improved during 6 treatment but relapsed after completed treatment (ClinicalTrials.gov identifier NCT01205464). In dermatology 6 and 7 were shown to be effective for the treatment of acne and rosacea, resulting in a significant reduction in inflammatory lesions.46 Two randomized, double-blind, placebo-controlled, multicenter trials have demonstrated the efficacy and excellent risk−benefit ratio of 6 (40 mg/day) in the treatment of rosacea.139 Bipolar disorder is characterized by reduced monoaminergic signaling and other neural changes. Such changes include dendritic remodeling, demyelination, and glial and neuronal cell loss deriving from chronic inflammation. Phase III clinical trial of 7 efficacy for bipolar depression treatment is currently ongoing. This trial has the specific aim of investigating the correlation between the expression levels of inflammatory cytokines and depression ratings in response to 7 (ClinicalTrials.gov identifier NCT01429272). In patients with adult and chronic periodontitis the oral administration of subantimicrobial doses of 6 also produced significant improvements in clinical parameters. These improvements include tooth attachment, probing depth, clinical attachment level, gingival index, plaque index, and decreased proinflammatory cytokines levels.140

fact, 7, which has a dimethylamino substituent at the C7 position of the D-ring, demonstrated superior scavenging activity in both lipid peroxidation and 2,2-diphenyl-1picrylhydrazyl (DPPH) assays with respect to the other TCs.49 The effective concentrations of TCs in cell-free assays were much higher (5−25 μM) than those providing protective effects in cell-based assays (1−20 nM). From this one gathers that direct antioxidant activity probably does not represent the primary mechanism of protection against oxidative injury to a cell.49 Therefore, several alternative mechanisms involving different intracellular pathways have been proposed. For example, in vitro and in vivo studies have shown that 7 and 6 can inhibit iNOS, which is responsible for the generation of excessive NO amounts in inflammatory and autoimmune processes.130,131 In murine macrophages and human cartilage affected by osteoarthritis, compounds 7 and 6 inhibit iNOS expression at the level of iNOS mRNA and protein expression. This implies a down-regulation of its specific activity.132 Another study suggests that the decrease in iNOS levels in LPS-stimulated cells treated with 7 was mediated by 7-induced TNF-α inhibition. Finally, implication of p38 MAPK pathway in the protective effect of TCs against oxidative stress has also been proposed.130 3. Anti-Inflammatory Effects. Acute inflammation is a temporary and self-limiting response to infection or injury. It ultimately serves to remove damaged tissue and generate new tissue. This results in restoration of tissue homeostasis and, subsequently, healing. By contrast, chronic inflammation, which is a result of immune dysregulation, plays a central role in the maintenance of pathological conditions. Chronic inflammation is characterized by an escalating cycle of tissue damage, resulting in autoimmunity, malignant transformation, or deleterious changes in tissue morphology and function.46 Proinflammatory cytokines, produced during inflammatory processes by activated immune effector cells, stimulate release of ROS and activation of MMPs. This results in direct injury to the surrounding healthy tissue. As previously discussed, TCs can both inhibit MMPs and protect from oxidative stress. Therefore, they are capable of exerting anti-inflammatory actions. In addition, it was found that TCs can reduce elevated levels of proinflammatory cytokines. Thus, in a murine model of LPS-induced lethal endotoxemia, TCs significantly reduced TNF-α and IL-1β levels in serum, producing a protective effect against LPSinduced shock.133 Since TCs did not affect directly the expression of cytokines by LPS-induced macrophages in vitro, the authors deduced that TCs likely modulated some upstream pathway implicated in the production of TNF-α and IL-1β.133 Conversely, Krakauer and Buckley demonstrated that 6 directly inhibited production of cytokines (IL-1β, IL-6, TNF-α, and IFN-γ) and chemokines (MCP-1, MIP-1α, and MIP-1β) by human peripheral blood mononuclear cells stimulated with staphylococcal exotoxins.134 In neuroinflammation, the effects of TCs are wide-ranging and include suppression of different inflammatory factors. These include TNF-α, cycloxygenase-2, interleukins (IL-2, IL-6, IL-1β), iNOS, MMPs, and kinases (apoptosis signaling kinase-1, c-jun N-terminal kinase, p38 mitogen-activated protein kinase).100 In vivo in a mouse model of induced acute or chronic colitis, treatment with 7 significantly reduced the levels of cytokines IL-1β, TNF-α, and IL-6 and iNOS expression in colonic tissues.131 Similarly, in a murine model of Kawasaki disease, associated with inflammatory response focused at the coronary M



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4. Antiapoptotic Effects. Apoptosis is a tightly regulated cell death program. It involves many factors and is important in maintaining tissue hemostasis. Activation of caspases is a key event in apoptotic cascades and may occur via extrinsic or intrinsic pathways. Extrinsic pathways are triggered by binding of death signal proteins to their cell surface receptor. This step is followed by activation of initiator caspase (caspase-8) which in turn activates downstream effector caspases. Intrinsic pathways can be activated by various types of intracellular and extracellular stress stimuli (for example, excessive intracellular levels of Ca2+). Such pathways involve permeabilization of the mitochondrial membrane and release of cytochrome c into the cytoplasm. Cytochrome c then forms a multiprotein complex known as the “apoptosome” and initiates activation of the caspase cascade through caspase-9.46,141 Caspase-independent apoptosis involves Smac/DIABLO (mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein binding protein with low pI) and apoptosis-inducing factor (AIF) proteins that are released from the intermembrane space into cytosol. TCs were shown to possess antiapoptotic activity, which is believed to involve the reduction of caspase expression.46 Thus, in a mouse model of HD, treatment with 7 resulted in downregulation of both caspase-1 and caspase-3.84 Neuroprotection by 7, associated with a reduction in caspase-1 and/or caspase-3 expression, has also been observed in animal models of traumatic brain injury, cerebral ischemia, and PD.46 The fact that 7 could not directly affect enzymatic activity of the caspases in the enzymatic assay84 indicates that there might be an upstream mechanism that results in caspase downregulation. One of the known physiological stimuli for caspase-9 and caspase-3 activation is cytochrome c release from mitochondria into the cytoplasm. In fact, several groups have reported that in rodent models of amyotrophic lateral sclerosis, HD, and spinal cord injury, 7 inhibited cytochrome c release from mitochondria. Besides that, it likely affected mitochondrial permeability transition (MPT) and reduced the expression levels of initiator caspases.142 In addition to cytochrom c, the decrease in release of other proapoptotic factors, such as AIF and Smac/Diablo in response to treatment with 7, has been observed both in vitro and in a mouse model of HD.142 Later, Antonenko et al. demonstrated that the effect of 7 was more likely related to the direct interaction of the drug with mitochondrial membrane in complex with Ca2+, resulting in formation of ion channels and prevention of excessive Ca2+ accumulation in the mitochondrial matrix.143 Moreover, the selective activity of TCs against mammalian cell mitochondria has also been previously described. Thus, it was shown that TCs not only selectively accumulate in mitochondria144 but also depolarize membrane potential, decrease respiratory control, and inhibit synthesis of mitochondrial DNA-encoded proteins.145 In fact, in human liver epithelial cells treatment with micromolar concentrations of 5, 6, and 7 decreased levels of a mitochondrial DNA-encoded protein (complex IV-subunit 1). They did not, however, decrease those of a nuclear DNAencoded protein (complex V-α subunit).146 As antibiotics, TCs target protein synthesis on bacterial ribosomes.61 Considering the structural similarity between bacterial ribosomes and eukaryotic mitochondrial ribosomes, it is not surprising that TCs can also selectively inhibit synthesis of mtDNA-encoded proteins. It is believed that impairment of mitochondrial biogenesis is also implicated in immunosuppressive properties of TCs. Mitogen-induced blasts formation requires ATP

generated in mitochondria. Since 6 is able to impair mitochondrial biogenesis, it inhibits blast formation and hence produces an immunosuppressive effect.147 TCs apparently exert the antiapoptotic activity by a mechanism involving mitochondrial stabilization. This mechanism ultimately results in a reduced release of cytochrome c and other proapoptotic proteins. At the same time, proapoptotic activity of 6 has been observed in human cancer cell culture. It was also associated with reduction of mitochondrial transmembrane potential and an increased cytochrome c release with attendant caspase-8 and caspase-9 activation.141 Curiously, the observed inhibitory effect on the growth of human and mouse cancer cells was observed for 6 but not for its analogue 7.148 Moreover, it was found that 6, toxic to human and mouse melanoma cells, did not affect normal cell growth of rat aortic vascular smooth muscle cells. This indicates that the effect of 6 may differ for normal and cancer cells.148 The mechanism of possible proapoptotic activity of 6 on cancer cells remains a point of investigation and study. The possible mechanisms that have been proposed include JNK activation mediated apoptosis148 and caspase independent mitochondrial apoptotic pathway.141

V. CONCLUSIONS Brown et al. first started to treat patients suffering from rheumatoid arthritis with 14 and described their beneficial effects in several papers.149 Although the mechanism of action of TCs was then unknown, there was general agreement on the possibility that their antimicrobial properties were responsible for the observed therapeutic effect. Subsequently, Paulus raised the question of whether various nonantimicrobial activities of TCs (such as MMPs inhibitory, anti-inflammatory, immunosuppressant) were involved in the clinical benefits in rheumatoid arthritis.150 Since then, ample evidence has been provided of the important role of TC nonantimicrobial activities in a variety of pathologies, suggesting that their protective effects could depend on a combination of pathological pathways.46,53,100,152 As discussed above, the pleiotropic chemical properties of TCs make them efficient against a variety of biological targets and underlie both nonantimicrobial and antimicrobial activities. To shed light on the possibility of distinguishing the therapeutic targets of TCs, we compared the treatment schedules and dosages used in different clinical trials. In almost all these trials the doses of TCs were very similar, with very few exceptions (acne, rosacea, and periodontal disease). The dosages generally used are 250−1000 mg/day for 5, 100−200 mg/day for 6, and 50−100 mg/day for 7. This is true for both short- and long-term treatments.45 The same dosages are generally recommended for the treatment of bacterial infections.45 Tetracycline blood levels at these dosages show dose related linearity, regardless of the pathological state of the patients.45 Two hours after a single oral dose of 100−200 mg of 6, peak serum concentrations range from 1.7 to 5.7 mg/L, falling to 1.45 mg/L at 24 h.45 After intravenous infusion of the same dose, peak plasma concentrations range from 5 to 10 mg/ L, falling slowly and persisting for 24 h at 1−2 mg/L.45 According to in vitro studies, 3−20 μg/mL concentrations of TCs, which can be achieved in patients after administration of 200 mg of the drug, are enough to exert MMPs inhibitory, antiinflammatory,130 antiapoptotic,151 and antioxidant49,121 activities. N



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Figure 3. Layout of the possible mechanism of action of tetracylines (rectangles) against a variety of pathogenic effects concurrent with amyloid deposition in amyloidoses (gray ellipses). The use of tetracyclines in amyloidoses therapy may affect the main pathological targets, such as aggregation and deposition of the misfolded proteins and ameliorate other pathological mechanisms associated with formation of amyloid deposits, including inflammation, ROS generation, apoptosis, and uncoupling of metal homeostasis. Tetracycline moieties known to display their therapeutic effect on each of these pathological events are indicated in bold. The absence of side chains R3 and R6 (dashed circles, metal chelation) increases MMPs inhibition.

In conclusion, considering the TCs doses given to patients for different pathologies, it is hard to establish specificity of the target affected by the drug. The final beneficial clinical outcome of TCs therapy is most likely a result of targeting multiple pathological events leading to a combination and/or synergy of effects (Figure 3). The use of TCs to treat amyloidoses may affect the main pathological target, such as aggregation and deposition of the misfolded proteins. They may also contribute to improving other pathological events, concurrent with amyloid deposit formation, including inflammation, ROS generation causing oxidative stress, apoptosis, and uncoupling of metal homeostasis. Our analysis of the literature indicates that there is very likely room for improving the therapeutic index of TCs in various diseases and a more rational approach in new treatment schedules would be welcome. Clinical trials need to be designed specifically to reassess the therapeutic activity of TCs, closely monitoring plasma levels in patients. On this basis only, it will be possible to rationally re-evaluate the potential of this family of molecules.

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Macromolecular Chemistry, University of Trieste, Trieste, Italy. At present she is attending the Advanced School of Applied Pharmacology at the Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy. She is involved in studies aimed at developing new peptides with antiamyloidogenic activity using structure and ligand base drug design. She also carries out structural analysis of amyloidogenic proteins by means of X-ray and neutron diffraction. Laura Colombo. Since 1985 she has been a Research Scientist in the Department of Molecular Biochemistry and Pharmacology at the Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy. Her research efforts focus on the development of physicochemical approaches for the identification of small molecules hindering the formation of soluble toxic oligomers and amyloid fibrils, with particular regard to prion-related encephalopathies and Alzheimer’s and Parkinson’s diseases. She has carried out the original preclinical studies that enabled doxycycline to enter phase II clinical studies as an antiamyloidogenic drug. Gianluigi Forloni. Since 2002, he has been the Head of the Department of Neuroscience at the Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy. His scientific interests are focused on the biological and genetic bases of aging-related disorders with particular regard to prion-related encephalopathies and Alzheimer’s and Parkinson’s diseases. He has investigated the role of protein misfolding in neurodegneration in experimental models to identify new pharmacological targets. His main achievements were obtained in the following fields: the identification of new pathogenic mutations in Alzheimer’s and Parkinson’s disease; the characterization of the apoptotic mechanism mediating the neurotoxicity of peptides derived from prion protein, β-amyloid, and α-synuclein; the development of animal models to investigate the pathogenesis of

AUTHOR INFORMATION

Corresponding Author

*Telephone: +390239014447. E-mail: mario.salmona@ marionegri.it. Notes

The authors declare no competing financial interest. Biographies Tatiana Stoilova received her Ph.D. from the Lomonosov Moscow State Academy, Russian Academy of Sciences, in 2001 followed by a postdoc at the Department of Biochemistry, Biophysics, and O



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2010 recommendations from the nomenclature committee of the International Society of Amyloidosis. Amyloid 2010, 17, 101−104. (3) Pepys, M. B. Amyloidosis. Annu. Rev. Med. 2006, 57, 223−241. (4) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fraser, P. E.; Pepys, M. B.; Blake, C. C. Common core structure of amyloid fibrils by synchrotron X-ray diffraction. J. Mol. Biol. 1997, 273, 729−739. (5) Kirschner, D. A.; Gross, A. A.; Hidalgo, M. M.; Inouye, H.; Gleason, K. A.; Abdelsayed, G. A.; Castillo, G. M.; Snow, A. D.; PozoRamajo, A.; Petty, S. A.; Decatur, S. M. Fiber diffraction as a screen for amyloid inhibitors. Curr. Alzheimer Res. 2008, 5, 288−307. (6) Dobson, C. M. Protein folding and misfolding. Nature 2003, 426, 884−890. (7) Hardy, J. A.; Higgins, G. A. Alzheimer’s disease: the amyloid cascade hypothesis. Science 1992, 256, 184−185. (8) Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigli, L.; Zurdo, J.; Taddei, N.; Ramponi, G.; Dobson, C. M.; Stefani, M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 2002, 416, 507−511. (9) Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 2003, 300, 486−489. (10) Glabe, C. G. Common mechanisms of amyloid oligomer pathogenesis in degenerative disease. Neurobiol. Aging 2006, 27, 570− 575. (11) Ferreira, S. T.; Vieira, M. N.; De Felice, F. G. Soluble protein oligomers as emerging toxins in Alzheimer’s and other amyloid diseases. IUBMB Life 2007, 59, 332−345. (12) Budka, H. Neuropathology of prion diseases. Br. Med. Bull. 2003, 66, 121−130. (13) Haass, C.; Selkoe, D. J. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid betapeptide. Nat. Rev. Mol. Cell Biol. 2007, 8, 101−112. (14) Cooper, G. J.; Aitken, J. F.; Zhang, S. Is type 2 diabetes an amyloidosis and does it really matter (to patients)? Diabetologia 2010, 53, 1011−1016. (15) Klein, W. L.; Krafft, G. A.; Finch, C. E. Targeting small Abeta oligomers: the solution to an Alzheimer’s disease conundrum? Trends Neurosci. 2001, 2, 219−224. (16) Zhao, H. L.; Lai, F. M.; Tong, P. C.; Zhong, D. R.; Yang, D.; Tomlinson, B.; Chan, J. C. Prevalence and clinicopathological characteristics of islet amyloid in Chinese patients with type 2 diabetes. Diabetes 2003, 52, 2759−2766. (17) Aitken, J. F.; Loomes, K. M.; Scott, D. W.; Reddy, S.; Phillips, A. R.; Prijic, G.; Fernando, C.; Zhang, S.; Broadhurst, R.; L’Huillier, P.; Cooper, G. J. Tetracycline treatment retards the onset and slows the progression of diabetes in human amylin/islet amyloid polypeptide transgenic mice. Diabetes 2010, 59, 161−171. (18) Bhak, G.; Choe, Y. J.; Paik, S. R. Mechanism of amyloidogenesis: nucleation-dependent fibrillation versus double-concerted fibrillation. BMB Rep. 2009, 42, 541−551. (19) Porter, M. Y.; Routledge, K. E.; Radford, S. E.; Hewitt, E. W. Characterization of the response of primary cells relevant to dialysisrelated amyloidosis to β2-microglobulin monomer and fibrils. PLoS One 2011, 6, e27353. (20) Stefani, M. Structural features and cytotoxicity of amyloid oligomers: implications in Alzheimer’s disease and other diseases with amyloid deposits. Prog. Neurobiol. 2012, 99, 226−245. (21) Merlini, G.; Bellotti, V. Molecular mechanisms of amyloidosis. N. Engl. J. Med. 2003, 349, 583−596. (22) Lashuel, H. A.; Hartley, D.; Petre, B. M.; Walz, T.; Lansbury, P. T., Jr. Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature 2002, 418, 291. (23) Huang, X.; Atwood, C. S.; Hartshorn, M. A.; Multhaup, G.; Goldstein, L. E.; Scarpa, R. C.; Cuajungco, M. P.; Gray, D. N.; Lim, J.; Moir, R. D.; Tanzi, R. E.; Bush, A. I. The Ab peptide of Alzheimer’s disease directly produces hydrogen peroxide through metal ion reduction. Biochemistry 1999, 38, 7609−7616.

neurodegenerative diseases; the identification of compounds hindering protein misfolding. Fabrizio Tagliavini. Since 2003, he has been the Head of the Department of Neurodegenerative Diseases and Director of the Division of Neuropathology−Neurology at the Istituto “Carlo Besta” Milan, Italy. He has been working in the field of neurodegenerative dementias associated with protein misfolding, in particular Alzheimer’s disease and prion-related encephalopathies with particular regard to the early stages of neuropathological changes and the biological properties of disease-associated proteins. His main achievements were obtained in the following fields: the identification of new pathogenic mutations linked to degenerative dementias; the identification and characterization of the early stages of neuropathological changes and new disease phenotypes; the development and characterization of relevant in vitro and in vivo models for pathogenesis studies; the identification of compounds that interact with misfolded proteins. Mario Salmona. Since 1997, he has been Head of the Department of Molecular Biochemistry and Pharmacology at the Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy. His research projects are focused on the structural and functional studies of specific, pharmacologically important gene products. Potential interactions between drugs and proteins were pursued at the molecular level by a variety of approaches ranging from animal studies to computer simulations. Particular attention was given to the relationship between protein misfolding and neurodegeneration. His main achievements were obtained in the following fields: identification and physicochemical characterization of a new pathogenic mutation in the Aβ peptide; biochemical, biophysical, and molecular modeling studies of neurotoxic peptides; identification of tetracyclines that specifically interact with misfolded proteins and inhibit amyloidogenesis.

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ACKNOWLEDGMENTS The critical reading of the manuscript by Dr. Vittorio Bertelè, Dr. Barbara La Ferla, and Dr. Enrico Monzani is gratefully acknowledged. This paper was supported by Banca Intesa Sanpaolo, Grant 2012-2013, and the Italian Ministry of Health, Grant RF-2009-1473239.

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ABBREVIATIONS USED 3D-QSAR, three-dimensional quantitative structure−activity relationship; AFM, atomic force microscopy; AIF, apoptosisinducing factor; β2M, β2-microglobulin; CJD, Creutzfeldt− Jakob disease; CMT, chemically modified derivative of tetracycline; CryAB, αB-crystallin; DPPH, 2,2-diphenyl-1picrylhydrazyl; FAP, familial amyloidotic polyneuropathy; FTIR, Fourier transform infrared; GSS, Gerstmann−Sträussler−Sheinken; HD, Huntington’s disease; hIAPP, human amylin/islet amyloid poypeptide; IDOX, 4′-iodo-4′deoxydoxorubicin; IVIg, intravenous immunoglobulin; LC, immunoglobulin light chain; MPT, mitochondrial permeability transition; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OPMD, oculopharyngeal muscular dystrophy; PABPN1, poly-(A) binding protein nuclear 1; PrP, prion protein; SAP, serum amyloid P component; TC, tetracycline; ThT, thioflavin T; TTR, transthyretin; TUDCA, tauroursodeoxycholic acid

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REFERENCES

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