Dendrimers as Potential Inhibitors of the Dimerization of the Capsid

Biomacromolecules 2010, 11, 2069–2078

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Dendrimers as Potential Inhibitors of the Dimerization of the Capsid Protein of HIV-1 Rosa Domeńech,† Olga Abian,‡,§ Rebeca Bocanegra,| Juan Correa,⊥ Ana Sousa-Herves,⊥ Ricardo Riguera,⊥ Mauricio G. Mateu,| Eduardo Fernandez-Megia,⊥ Adriań Vela´zquez-Campoy,*,‡,# and Jose´ L. Neira*,†,‡ Instituto de Biologıá Molecular y Celular, Universidad Miguel Hernańdez, Elche, Alicante, Spain, Instituto de Biocomputacioń y Fı´sica de Sistemas Complejos, Universidad de Zaragoza, Spain, I+CS (Aragon Health Sciences Institute), CIBERehd, Zaragoza, Spain, Centro de Biologıá Molecular Severo Ochoa (CSIC-UAM), Universidad Autońoma de Madrid, Cantoblanco, Madrid, Spain, Departamento de Quimica Orgańica, Facultad de Quı´mica, and Unidad de RMN de Biomolećulas Asociada al CSIC, Universidad de Santiago de Compostela, Santiago de Compostela, La Corunã, Spain, and Fundacioń ARAID, Diputacioń General de Aragoń, Zaragoza, Spain Received April 21, 2010; Revised Manuscript Received June 9, 2010

Assembly of the mature human immunodeficiency virus type 1 capsid involves the oligomerization of the capsid protein, CA. The C-terminal domain of CA, CTD, participates both in the formation of CA hexamers and in the joining of hexamers through homodimerization. Intact CA and the isolated CTD are able to homodimerize in solution with similar affinity (dissociation constant in the order of 10 µM); CTD homodimerization involves mainly an R-helical region. In this work, we show that first-generation gallic acid-triethylene glycol (GATG) dendrimers bind to CTD. The binding region is mainly formed by residues involved in the homodimerization interface of CTD. The dissociation constant of the dendrimer-CTD complexes is in the range of micromolar, as shown by ITC. Further, the affinity for CTD of some of the dendrimers is similar to that of synthetic peptides capable of binding to the dimerization region, and it is also similar to the homodimerization affinity of both CTD and CA. Moreover, one of the dendrimers, with a relatively large hydrophobic moiety at the dendritic branching (a benzoate), was able to hamper the assembly in vitro of the human immunodeficiency virus capsid. These results open the possibility of considering dendrimers as lead compounds for the development of antihuman immunodeficiency virus drugs targeting capsid assembly.

Introduction Trying to inhibit protein-protein interactions with a small ligand for therapeutic purposes is an attractive, but challenging idea. This is probably due to the relatively large and stereochemically complex protein-protein interfaces that make it difficult for a small molecule to encompass the whole binding site.1,2 Entropic considerations may also aggravate the problem of achieving enough affinity and specificity between the inhibitor molecule and the protein surface. Recently, however, specifically designed small molecules have been shown to disrupt such large and complex interfaces by binding to “hotspots”.2 A promising example is that of the B-cell lymphoma protein (Bcl-XL) and the pro-apoptotic molecule Bcl-2 antagonist of cell death (BAK), currently in phases 1 and 2 clinical trials.3 Virus capsids provide other biomedically relevant targets for the design of proteinprotein interfacial inhibitors. Because of the omnipresent nature of protein-protein interactions in virion assembly and maturation, it could be possible to design antiviral strategies based on the inhibition of those macromolecular complexes.4 * To whom correspondence should be addressed. Tel.: +34 976762996 (A.V.-C.); +34 966658459 (J.L.N.). Fax: +34 976762990 (A.V.-C.); +34 966658758 (J.L.N.). E-mail: [email protected] (A.V.-C.); [email protected] (J.L.N.). † Universidad Miguel Hernańdez. ‡ Universidad de Zaragoza. § I+CS (Aragon Health Sciences Institute). | Universidad Autońoma de Madrid. ⊥ Universidad de Santiago de Compostela. # Fundacioń ARAID.

Human immunodeficiency virus (HIV), the agent responsible for AIDS, belongs to the retrovirus family. The HIV genome is a model of economic packaging because the virus uses complex processing (both in the production and cleavage of mRNAs and in the final viral polypeptides) to generate several proteins that can work in the host cell. Among them, one of the most fascinating examples is perhaps the Gag-polyprotein. Assembly of the immature HIV type 1 (HIV-1) capsid occurs through the controlled polymerization of the Gag-polyprotein, which is transported to the plasma membrane of infected cells, where morphogenesis of the immature, noninfectious virion occurs. Immediately after budding of the immature virion, maturation is initiated by the cleavage of the Gag protein by a viral protease, which yields the MA (matrix), CA (capsid), NC (nucleocapsid), and p6 proteins, as well as two spacer peptides. This maturation process induces conformational changes in CA, which reassembles as an independent protein to form a capsid with a distinctive conical shape.5,6 Because of its critical role during HIV-1 morphogenesis, CA has emerged as a promising target for the development of new anti-HIV drugs based on disruption of capsid assembly.7,8 The CA protein is formed by two independently folded domains: the N-terminal domain, NTD, and the C-terminal domain, CTD, separated by a flexible linker.9-12 The NTD (residues 1-146 in the numbering of the whole intact protein) is composed of five coiled-coil R-helices (corresponding to helices 1-5 of CA), with two additional short R-helices (helices 6 and 7) following an extended proline-rich loop. The CTD

10.1021/bm100432x  2010 American Chemical Society Published on Web 07/13/2010

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Figure 1. Crystal structure of the wild-type CTD dimer. The monomers are depicted in the same color (blue) and the dimerization helix is highlighted (gold), with the side chain of the sole trypotophan (Trp184) in each monomer indicated by sticks. The figure was produced using Pymol (http://www.pymol.org)73 by using the Protein Data Bank file for CTD (accession no. 1A43).10 The different R-helices are indicated; the numbering of these helices corresponds to helices 8-11 of the whole intact CA.

domain (corresponding to residues 147-231) is a dimer both in solution and in the crystal form.10,13 Each CTD monomer is composed of a short 310-helix followed by a strand and four R-helices (helices 8-11 of CA): R-helix 8 (residues 160-172), R-helix 9 (residues 178-191), R-helix 10 (residues 195-202), and R-helix 11 (residues 209-214), which are connected by short loops or turn-like structures (Figure 1). At the beginning of CTD, there is a polypeptide region (Asp152-Leu172), called the major homology region (MHR), which is highly conserved among retroviruses and whose functions have not been fully elucidated. The dimerization interface of CTD is mainly formed by the mutual docking of R-helix 9 from each monomer, with the side chains of each tryptophan (Trp184) deeply buried in the dimer interface. The two additional aromatic residues in each CTD monomer, Tyr164 and Tyr169, are located in the hydrophobic core of each monomer, far away from the dimer interface. The dissociation constant of CTD is 10 ( 3 µM,10,14 similar to that of intact CA, 18.0 ( 0.6 µM;10,14 then, the CA dimerization interface is fully contained within the isolated CTD. This observation has paved the way to the search for inhibitors of HIV-1 assembly, because molecules able to dissociate CTD dimers in solution may be considered as potential inhibitors of CA assembly and HIV-1 infectivity (although this assumption must be always tested by direct experiments). The first molecules specifically targeted against the self-association of CTD, or even the CTD-NTD interface (which is also important during capsid assembly6), have involved (i) peptides mimicking the sequence of the dimerization interface;15 (ii) peptides selected from a combinatorial library by phage display;16 and (iii) peptides based on those selected by phage display techniques, which have been further redesigned to increase the helical content.17 This latter design is able to penetrate cells and hampers HIV-1 infectivity, although it does not abolish CTD dimerization completely.17 In this work, we go a step further by testing dendrimers to inhibit CTD dimerization and HIV-1 capsid assembly in vitro. Other organic molecules have been shown to inhibit CA assembly, but instead of targeting the CTD dimerization interface, they interact with the oligomerization interfaces of NTD.18

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Dendrimers are highly branched, synthetic macromolecules with several terminal groups emanating from a central core. They are synthesized through a stepwise controlled process where new shells of branching points (generations) provide additional terminal groups on the surface.19,20 Dendrimers have attracted significant interest in supra-macromolecular chemistry due to the high degree of control over their dimensions.21 They are flexible, and contrary to traditional polymers, dendrimers are monodisperse. Furthermore, dendrimers can be exploited for biomedical purposes, such as drug and gene delivery, and for the interaction with biological macromolecules and biostructures.19,22,23 In addition, dendrimers can display therapeutic activities by exploiting their inherent capacity to multivalently interact with biological receptors. In this way, dendrimers with microbicide24-26 and anti-inflammatory20 activities, as well as dendrimers with the ability to activate human monocites and stimulate the growth of human natural killer cells,27,28 have been recently described; further, dendrimers as heparin mimetics29,30 and immunomodulatory and antiangiogenic dendrimers31 have been reported. There are also reports on the use of dendrimers as polymer therapeutics to dissolve prion protein aggregates and to hamper fibril formation of prion and Aβ peptides.32-39 In this context, it is worth mentioning the mimicry of dendrimers to protein subunits or protein domains in their ability to self-assemble and to produce supramolecular structures.39 We have hypothesized that if dendrimers can destabilize the tertiary structure of proteins, they could also be used to destabilize the quaternary structure of CTD and then CA assembly to form viral capsid. In this work, we analyze the ability of GATG (gallic acid-triethylene glycol) dendrimers to disrupt the quaternary structure of CTD (from now on, we will call the dendrimers as [Gn]-X, being “n” the dendrimer generation and “X” the terminal group, Scheme 1). The results with various dendrimer generations and different groups at their highly branched surface suggest that only [G1] dendrimers were bound to the dimerization region, or nearby residues, of CTD. The dissociation constants of GATG-dendrimer-CTD complexes were comparable to the self-dissociation constant of CTD (10 ( 3 µM) and very similar to those of designed peptides weakening CTD dimerization.7,15,16 However, only the molecule with a benzoate moiety at the dendrimer branching was able to hamper assembly of the HIV-1 capsid in vitro. At the best of our knowledge, these studies are the first to suggest dendrimers as possible inhibitors of the dimerization of CTD and of the assembly of the HIV-1 capsid.

Experimental Section Materials. Deuterium oxide was obtained from Apollo Scientific (Stockport, U.K.), and the sodium trimethylsilyl [2,2,3,3-2H4] propionate, TSP, was obtained from Sigma (St. Louis, MO). Dialysis tubing with a molecular weight cutoff of 3500 Da was from Spectrapor (Spectrum Laboratories, Ibaraki, Japan). Standard suppliers were used for all other chemicals. Water was deionized and purified on a Millipore system (Millipore, MA). Design and Synthesis of the Dendrimers. The functionalized GATG dendrimers of this study were prepared as described.40-42 Briefly, our approach involves the peripheral decoration of azideterminated dendrimers by means of the Cu(I)-catalyzed azide-alkyne [3 + 2] cyclo-addition (CuAAC). Alternatively, the reduction of the terminal azides leads to amino-functionalized dendrimers. Three different types of dendrimers (Scheme 1) were assayed for possible binding to CTD. We tried the first ([G1]) and second generations ([G2]) of [Gn]-OSO3Na and [Gn]-Lac to map for the influence of the dendrimer generation and the nature of the terminal

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Scheme 1. Diagram Showing the Different Dendrimers with Their Corresponding Surface Functionalization Used in This Worka

a The molecular masses of the compounds are as follows: PEG-[G1]-OSO3Na, 6340.3 g/mol; [G1]-Man, 1289.3 g/mol; [G1]-NH2, 604.73 g/mol; [G1]SO3Na, 2034.31 g/mol; [G1]-CO2Na, 1209.36 g/mol; [G1]-Lac, 1847.6 g/mol; [G1]-OSO3Na, 1199.1 g/mol; [G2]-Lac, 5921.7 g/mol; and [G2]-OSO3Na, 4023.8 g/mol.

dendritic ligands (ionic (sulfate) or neutral (β-D-lactose)) on the binding to CTD. The second series involved only G1 dendrimers carrying different terminal groups: primary amine ([G1]-NH2), a highly hydrophobic sulfonate group ([G1]-SO3Na), a benzoate group ([G1]-CO2Na), and the monosaccharide R-D-mannose ([G1]-Man; Scheme 1). And the third approach involved the use of a sulfated PEG-dendritic block copolymer of G1, carrying a poly ethylene glycol chain of molecular weight 5000 Da attached to the focal point of the dendritic block (PEG[G1]-OSO3Na). PEG is recognized as the hydrophilic polymer of choice for the functionalization of polymers and nanostructures for applications in drug delivery and biomedicine due to its low toxicity and immunogenicity and its high biocompatibility.43,44 Its incorporation at the focal point of GATG dendrimers is intended to confer them further solubility and biocompatibility, as well as better biodistribution. Protein Expression and Purification. The wild-type CTD protein was purified as described.45,46 The 15N-labeled CTDW184A, monomeric mutant protein was obtained by using Bioexpress medium (Cambridge Isotope Laboratories, Andover, MA) using the same protocol described.47 The intact CA protein was expressed and purified as described.48 Protein stocks were run in SDS-PAGE gels and found to be >97% pure. Protein concentration was determined from the absorbance of individual amino acid compounds.49 Fluorescence. Spectra were collected on a Cary Eclipse spectrofluorometer (Varian, California) interfaced with a Peltier system. A 1 cm path length quartz cell (Hellma) was used. Experiments were performed at 25 °C in phosphate buffer at pH 7.0 (50 mM) with equimolar amounts of dendrimer and wild-type CTD (20 or 200 µM). (a) Steady State Fluorescence Measurements. Spectra were acquired by excitation at 280 or 295 nm; the emission fluorescence was collected between 300 and 400 nm. The excitation and emission slits were set

to 5 nm; the response was 1 nm. All the isolated GATG dendrimers had fluorescence properties (see Results). (b) Thermal Denaturation. Analysis of thermal denaturation and its reversibility was carried out as described by excitation at 280 or 295 nm.50 Emission fluorescence was collected at 315, 335, and 350 nm; the scan rate was 60 °C/h, and the data were acquired every 0.2 °C. Thermal denaturations were reversible in all complexes; the isolated dendrimers did not show a sigmoidal transition when heated (data not shown). Circular Dichroism. Spectra were collected on a Jasco J810 (Japan) spectropolarimeter with a Peltier unit. The instrument was periodically calibrated with (+)-10-camphorsulfonic acid. Spectra were acquired at 25 °C in phosphate buffer at pH 7.0 (50 mM). Far-UV measurements were performed using equimolar amounts of wild-type CTD and the corresponding dendrimer (20 or 200 µM) in 0.1 cm path length quartz cells (Hellma). (a) Steady State Measurements. Spectra were acquired with a response time of 2 s and averaged over six scans, with a scan speed of 50 nm/min. The step resolution was 0.2 nm, and the bandwidth was 1 nm. Isolated dendrimers did not show a far-UV CD spectra (data not shown), as observed in other dendrimers.51 (b) Thermal Denaturation. Thermal denaturations were carried out at 60 °C/h, with a response of 8 s, and by following the changes in ellipticity at 222 nm. All the transitions were reversible. The isolated dendrimers did not show a sigmoidal transition when heated (data not shown). Isothermal Titration Calorimetry. ITC measurements were performed by using a VP-ITC isothermal titration calorimeter (MicroCal, Northampton, MA). Measurements were carried out at 25 °C in Tris or phosphate buffer 12 mM (pH 7.0). A sample cell (1.4 mL) was loaded

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with wild-type dimeric CTD at 20 µM; the corresponding dendrimer was loaded into the syringe at a concentration of 400 µM. A total of 28 injections of 10 µL were added sequentially to the sample cell after 400 s spacing to ensure that the thermal power returned to the baseline before the next injection. The amount of power required to maintain the reaction cell at constant temperature after each injection was monitored as a function of time. The isotherms (differential heat upon binding versus the molar ratio: [dendrimer]/[wild-type CTD]) were fitted to a single-site model assuming that the complex with wild-type CTD had a 1:1 stoichiometry. Data were analyzed with software developed in our laboratory, implemented in the software package Origin 7.0 (OriginLab). As a control experiment, the individual dilution heats for the intact dendrimers were determined under the same experimental conditions by carrying out identical injections of the organic molecule into the sample cell, which contained only buffer. NMR Spectroscopy. The NMR experiments were acquired at 25 °C on a Bruker Avance DRX-500 spectrometer (Bruker GmbH) equipped with a triple resonance probe and z-pulse field gradients. TSP was used as the internal chemical shift reference in the homonuclear experiments.15 N chemical shifts of monomeric CTDW184A were referenced following the method of Wishart and co-workers.52 All experiments were carried out at pH 7.0, 50 mM phosphate buffer. (a) 2D 15N-HSQC-NMR Spectra. All spectra were acquired in the phase-sensitive mode. Frequency discrimination in the indirect dimensions was achieved by using the Echo/Antiecho-TPPI method. The 2D15 N-HSQC experiments53 were acquired with 4 K data points in the 1 H dimension, and 200 scans in the 15N axis. The spectral widths were 15 and 35 ppm in the 1H and 15N dimensions, respectively. Water was suppressed with the WATERGATE sequence.54 The concentration of the CTDW184A protein was 120 µM in all cases, and the corresponding dendrimer concentration was four to five times higher. We used the monomeric mutant CTDW184A protein, instead of the wild-type dimeric CTD protein, to avoid problems with signal broadening of the residues involved in the dimerization interface;14,55 thus, when using the monomeric CTDW184A, we have followed the same reasoning as other authors mapping the peptide-binding regions of CTD.16,17 In turn, this will facilitate the comparison among the results with the different systems. Assignments of the CTDW184A in the HSQC spectra were those previously reported.47,56 To allow for a comparison among the different dendrimers and to account for errors in protein concentration, signal intensity of each peak in the HSQC spectra was calculated by using XWINNMR software and calibrated with respect to the intensity of Leu231 (the C-terminal residue of CTD). (b) Measurements of the T2 (TransVerse Relaxation Time). Measurements of the T2 provide a convenient method to determine the molecular mass of a macromolecule, M, and its complexes. We measured the T2 of the dendrimer-CTDW184A complexes by using the 1-1 echo sequence (at echo times of 2.9 ms and 400 µs).57 Then, because the correlation time, τc, is τc ≈ (1)/(T2)58 and, roughly, M is twice the τc, the mass of the biomolecule or its complexes can be estimated. The width of Gly156 (the most downfield shifted signal) was measured in the echo experiments. Given the errors inherent to the technique,58 and the M of the complexes, we estimated that only variations larger than 10% in the measured T2 of the complex when compared to that of the isolated CTDW184A are significant (Scheme 1). Kinetic Analysis of CA Polymerization In Vitro. Polymerization of recombinant CA into capsid-like structures requires molar concentrations of NaCl59,60 or, alternatively, high concentrations of a macromolecular crowding agent (such as Ficoll) at physiological salt concentrations.48 Addition of both high salt and Ficoll concentrations leads to a substantial increase in the amount of assembled capsids. Assembly reactions for this study were carried out at 2.25 M NaCl, pH 7.4 (50 mM, sodium phosphate buffer), and 46 µM CA, either in the absence or presence of 100 g/L Ficoll 70 as a macromolecular crowding agent. A volume of CA solution at the appropriate concentration in phosphate buffer was introduced into a spectrophotometer cuvette (10 × 2 mm of internal section). The assembly reaction was triggered

Domeńech et al. by adding a solution containing either 4 M NaCl in 50 mM phosphate buffer at the specified pH or 4 M NaCl and 179 g/L Ficoll-70 in 50 mM phosphate buffer to get the final concentrations desired for each component. For inhibition assays, CA was mixed with the appropriate amounts of dendrimers and incubated for 30 min before triggering the reaction as indicated above. The components in a final volume of 500 µL were rapidly mixed by repeated inversion of the cell. The pH of the final reaction mixture was checked in a test sample. The timedependent increase in the optical density at 350 nm as a measure of the light scattered by the assembled particles was monitored at 25 °C using a Shimadzu UV-1603 spectrophotometer, with data points collected every 6 s. Traces of the variation in the turbidity were analyzed as described previously.48

Results Dendrimers Interact with the Wild-Type, Dimeric CTD. To detect the interaction between dendrimers and CTD, we followed a three-part approach. First, we mapped the differences in the fluorescence or CD spectra upon complex formation; the presence of Trp184 in the CTD dimerization interface (Figure 1) allowed us to map for changes in the protein structure. Second, if there was binding between wild-type CTD and the dendrimer, then the thermal denaturation of isolated wild-type CTD would be different (in shape and in Tm) to that observed in the presence of the dendrimer.61 Binding of the dendrimer to the native state of wild-type CTD, in the absence of binding to the denatured state, will necessarily lead to an increase in the melting temperature; conversely, binding to the unfolded state of CTD, in the absence of binding to the native state, will decrease the melting temperature relative to the protein in the absence of ligand.62 Finally, we measured the T2 transverse relaxation time of the dendrimer-CTDW184A complexes, and we compared the values to that of the isolated monomeric CTDW184A. The isolated dendrimers showed an intense fluorescence emission spectra (either by excitation at 280 or 295 nm; Figures 2A,B). The fluorescence spectra of the equimolar dendrimerCTD complexes were different to the addition spectra obtained by the sum of the isolated spectra of dendrimer and wild-type CTD. Especially dramatic were the differences observed in the fluorescence spectra for the sulfonate ([G1]-SO3Na) and the benzoate groups ([G1]-CO2Na); in both examples (Figure 2A), there is a strong quenching of the fluorescence of Trp184 and of the aromatic moiety of the dendrimer (Scheme 1) upon binding, suggesting the proximity of the sole tryptophan to the aromatic moiety at the dendrimer branching. Because the tryptophan is deeply involved in the dimerization interface of CTD,10 these results suggest that dendrimers bind at, or closed to, that region. The only exceptions to the changes in fluorescence spectra of CTD upon binding were the [G2] dendrimers, namely, [G2]-OSO3Na and [G2]-Lac, where the sum spectra and those of the complexes were identical. Thus, it seems that the branched structure of the [G2] series hampers binding to wild-type CTD. Steady-state far-UV spectra of the complexes did also show differences with the sum spectra and that of wild-type CTD (Figure 3A, open triangles). Three dendrimer groups can be established according to their behavior in the CD spectra upon binding: (i) those associated with a large increase of the ellipticity (in absolute value) in the spectrum of the dendrimerCTD complex when compared to that of isolated CTD (dendrimers [G1]-NH2, [G1]-CO2Na, [G1]-SO3Na, and PEG-[G1]OSO3Na; Figure 3A, black line); (ii) those associated with smaller changes in ellipticity upon complex formation (den-


Figure 2. Binding between the dendrimers and wild-type, dimeric CTD, as monitored by fluorescence. (A) Steady-state fluorescence spectra for [G1]-SO3Na (filled symbols and dotted line) and [G1]CO2Na (open symbols and continuous line). Fluorescence excitation was carried out at 280 nm; protein/dendrimer concentration was 200/ 200 µM. (B) Steady-state fluorescence spectra for [G1]-NH2 (open symbols and continuous black line), PEG-[G1]-SO3Na (filled symbols and dotted black line), and [G1]-Man (filled symbols and continuous blue line). Fluorescence excitation was carried out at 280 nm, and protein/dendrimer concentration was 20/20 µM. In panels A and B, the spectrum of isolated wild-type CTD is also indicated to allow for a comparison; further, the spectra of the complex and those obtained by adding the spectra of each reactant are also indicated. (C) Dendrimer-CTD interactions followed by thermal denaturations: isolated CTD (open circles); [G1]-SO3Na (open squares); [G1]-CO2Na (open diamonds); and [G1]-OSO3Na (filled circles). Fluorescence excitation was at 295 nm, and emission was collected at 350 nm; the protein/dendrimer concentration was 20/20 µM.

drimers [G1]-OSO3Na, [G1]-Lac, and [G1]-Man; Figure 3A, red line); and (iii) dendrimers that upon addition to CTD did not alter the ellipticity of the spectrum of the isolated protein (dendrimers [G2]-OSO3Na and [G2]-Lac; data not shown). When changes were observed, there was always an increase in the ellipticity (in absolute value), suggesting that upon binding the helicity of CTD was increased. These findings differ from results by other groups who found that dendrimer binding induces partial unfolding of proteins (that is, decrease of the ellipticity or even the reduction of the catalytic activity).51,63,64 On the other hand, thermal denaturations followed either by fluorescence (Figure 2C) or CD (Figure 3B) did not show a clear change in the Tms, but the shape of the sigmoidal curves became broader in the presence of the dendrimers. Moreover, the most important changes in the shape of the sigmoidal curve were observed for the [G1]-SO3Na-CTD complex, followed by those in the [G1]-CO2Na-CTD one (Figure 2C). The absence of variations in the Tm could be due to the fact that the stabilization induced by the ligand (and given by +RTln(1 + [L]/KD)) is not high enough.


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Figure 3. Binding between the dendrimers and wild-type, dimeric CTD as monitored by far-UV CD. (A) The steady-state spectra of the complex (in squares) and the spectra obtained by adding the corresponding ones of each reactant (circles). The spectra of [G1]CO2Na are indicated in black and open symbols; those of [G1]OSO3Na in red color and filled symbols. The spectrum of wild-type CTD is also shown for comparison (open triangles, black color). (B) Dendrimer-CTD interactions followed by thermal denaturations at 222 nm: isolated CTD (red, open circles), [G1]-CO2Na (black, open squares), and [G1]-OSO3Na (black, open diamonds); the scale on the y-axis is arbitrary. The protein/dendrimer concentration in the steady-state and thermal denaturations was 20/20 µM. Table 1. Dissociation Constant of Dendrimers to Wild-Type Dimeric CTD and Measurements of the T2 Relaxation Time of the Complexes Formed with Monomeric CTDW184Aa

dendrimer [G1]-NH2 [G1]-Lacc [G1]-OSO3Na PEG-[G1]-OSO3Na [G1]-Man [G1]-SO3Nac [G1]-CO2Na CTDW184A

KD (µM)b 8.3 1.3 5.2 303 7.7

T2 relaxation time (ms; molecular mass, kDa) 42.0 (9.6) 40.0 (10.0) 40.0 (10.0) 24.4 (16.4) 36.5 (10.9) 42.6 (9.1) 30.1 (13.2) 42.9 (9.3)

a The determined T2 relaxation time of monomeric CTDW184A is also shown. Within parentheses for each T2 relaxation measurement is the estimated molecular mass (see Experimental Section). b Determined at 25 °C by ITC. c The affinity could not determined.

Finally, we also tried to detect dendrimer binding by measuring the T2 relaxation time of the 1H in the GATGdendrimer complexes. We should expect that as the molecular weight increases upon binding, the T2 relaxation time of the resulting complex would be smaller. Taking into account the limitations of the technique (with errors in the range of 10%), only the T2 relaxation times of [G1]-CO2Na, PEG-[G1]OSO3Na, and [G1]-Man are significantly different from those of monomeric CTDW184A (Table 1), suggesting binding of those compounds. It could be thought that the similarity between the T2 relaxation-time of complexes and that of monomeric CTDW184A is due to the fact we are using the monomeric

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Figure 4. Binding between dendrimers and wild-type dimeric CTD as determined by ITC measurements. Binding of wild-type CTD to [G1]-NH2 showing the raw data (top panel) and the binding curve (low panel). The inset shows the binding curve for the PEG-[G1]OSO3Na. Measurements were carried out at 25 °C in 12 mM Tris (pH 7.0).

species; however, control experiments of T2 measurements using wild-type dimeric CTD and selected dendrimers also failed to reveal significant differences (data not shown). To conclude, the [G1] dendrimers are able to interact with the wild-type CTD, but the larger [G2] dendrimers explored in this work failed to do so, perhaps due to steric restrictions. Measurement of the Affinity Constant of the Dendrimers for the Wild-Type, Dimeric CTD. Thermal denaturation analyses (followed by CD and fluorescence) and steady state fluorescence and CD provided unambiguous evidence of an interaction between dendrimers and wild-type CTD. Next, we determined the affinity by using ITC (Table 1, Figure 4). The dissociation constants are most of them in the range from 1 to 10 µM, and only that of [G1]-Man showed a larger value (300 µM). We were not able to measure the affinity constant of [G1]SO3Na or [G1]-Lac, probably due to a small binding affinity and/or a small binding enthalpy. Determining the Dendrimer Binding Site in CTD. The above biophysical probes have shown that there is binding between the dendrimers and the dimeric wild-type CTD, but what is the region of the protein involved in binding? We tried to map the binding site of dendrimers in CTD by using 2D15 N-HSQC spectra of monomeric CTDW184A. In this type of spectrum, main chain amide protons are observed with one signal for each amino acid. The use of the mutant monomeric protein will allow (i) an easier comparison with results of peptides that are bound to monomeric mutants of CTD16,17 and (ii) an easier assignment of the residues which are modified by the binding, because the wild-type CTD protein shows severe signal broadening due to the dimerization equilibrium.14,55,56 When dendrimers were added to a solution of CTDW184A, it was found that most of the peaks did not change their chemical shifts. However, there were several resonances whose intensities changed in the presence of the dendrimer, and then they were

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broadening upon binding to dendrimer. The variation of the signal intensity for a backbone amide 1H-15N cross-peak in the HSQC spectrum indicates that a population of the CTD protein is engaged in dendrimer binding in a way where the amide group of this residue exhibits broader lines. Furthermore, the absence of changes in chemical shifts and a large signal broadening suggest that the exchange kinetics of the complex formation equilibria are slow on the NMR time scale; similar findings (that is, broadening of signals, but absence of large change in chemical shifts) have been observed in other binding equilibria followed by NMR.65 Table 2 shows a dendrimer-by-dendrimer analysis, indicating the peaks whose signal intensities changed in the presence of the organic molecule (residues whose HSQC signal intensity was found to vary in more than two of the assayed dendrimers are shown in bold). It is interesting to note that (i) PEG-[G1]-OSO3Na showed the largest changes in peak intensity, and (ii) we were able to see a change of 0.1 ppm in the amide proton chemical shift of Tyr169 in [G1]-SO3Na. Residues that appear to be more frequently involved in the binding of the dendrimers are highlighted on the structure of the monomeric species of the protein47,56 (Figure 5). Those residues are located in helix 9, the dimerization helix (residues Ser178-Asn193), which does not appear to be wellformed in the isolated monomer.47,66 The MHR region (residues Asp152-Leu172) also seems to be a preferred polypeptide patch for binding. The rest of the residues appear located at the C-cap of helix 11 and the in-between loop connecting the last two helices. It could be thought that the dendrimer binding mode of the mutant monomeric species could be different to that of the wild-type dimeric one; however, and although we cannot rule out completely that possibility, several pieces of evidence suggest that binding in both CTD species is similar. First, X-ray and NMR16,67,68 studies on peptides, which bind to the dimerization interface of CTD, show that the recognition regions, for those residues whose signals in the HSQC spectrum of the dimeric CTD species could be observed, are identical. Second, we carried out binding control experiments with the dimeric CTD and two selected dendrimers (PEG-[G1]-OSO3Na and [G1]CO2Na) and we observed (within the poorer signal intensity of the spectra when compared to those for monomeric species) that for the signals of some residues, which are not excessively broadened and are well-separated from the rest of the amide protons (such as Asp152 and Phe161), variations in the intensity could be also observed (data not shown). And finally, we believe that it would be very unlikely that, whereas the tryptophan residue in dimeric wild-type is involved in binding (as suggested by the fluorescence experiments, see above), the nearby residues (as those mapped in the NMR experiments in the monomeric species) did not intervene. As expected, in a control experiment with [G2]-Lac, changes in peak intensity were observed in a sole residue, which is located far from the dimerization helix (Table 2). Finally, from the above results, it is clear that dendrimers showing the largest number of affected peak intensities are those containing a larger hydrophobic moiety, namely, [G1]-CO2Na and [G1]-SO3Na (Table 2). To summarize, some residues of the dimerization helix appear to form an important part of the regions of CTD involved in binding to most of the tested dendrimers. Testing the Inhibitory Activity of Dendrimers on an In Vitro Model for Assembly of the HIV-1 Capsid. Positive binding of the [G1] dendrimers at, or close to, the CTD

Cys218

Only residues whose changes in intensity were larger than 10 units of the corresponding cross-peak in the spectrum of isolated CTDW184A are indicated. The different regions of the protein are those described by X-ray in the structure of each monomer in wild-type CTD.10 Residues in bold are those whose signal intensity appears changed in more than two dendrimers. Spectra were acquired at 25 °C. b This residue overlaps with Ser149 and His226. c This residue overlaps with Leu151. d This residue overlaps with Glu178. e This residue overlaps with Glu175 and Glu213. f This residue overlaps with Lys227. g The mutated residue in CTDW184A.

a

Thr210, Glu213 Ala217

in-between loop helix 10 (195-202) in-between loop helix 11 (209-214) C-terminus

Glu180b, Val181, Asn183, Thr186, Thr188, Leu190c Asn193d Lys199

Thr210 Gly225

Cys218

Thr210 Cys218, Val221, Gly225

Lys203 Thr210, Met214 Cys218, Gly220, Gly225 Asp197 Lys203, Leu205

Asn193d

Asn183, Ala184g, Met185 Glu180b, Lys182f, Asn183, Thr186, Thr188, Leu190c

Asp152, Phe161, Arg162, Asp166, Phe168, Lys170, Leu172

N-terminus MHR (152-172) and R-helix 8 (160-172) in-between loop Helix 9 (178-191)

Lys170, Arg173

Ile153, Arg154, Gln155, Glu159, Asp163 Gln176e Glu180b, Lys182f, Ala184g

Lys182f, Asn183, Thr186, Thr188, Val191

Ile150 Arg154 Thr148 Lys158

[G1]-Man [G1]-SO3Na dendrimer [G1]-CO2Na PEG-[G1]-OSO3Na [G1]-NH2 protein region

Table 2. Residues of CTDW184A Whose Intensities in the HSQC Spectra Were Changed upon Dendrimer Additiona


Lys199 Lys203 Met214 Ala217

Ala184g, Met185, Leu190

Ile150

[G2]-Lac

[G1]-Lac


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Figure 5. Binding of the GATG dendrimers to monomeric CTDW184A. The NMR structure of the monomeric mutant CTDW184A is indicated. The residues in red are those that are observed more frequently to change their peak intensities in the presence of the GATG dendrimers (in bold in Table 2). The figure was produced using Pymol (http:// www.pymol.org)73 using the Protein Data Bank file for CTDW184A (accession no. 2JO0).47 The helix 9, which comprises the majority of the dimer-dimer interface in wild-type CTD, is not fully formed most of the time in the monomer.

dimerization interface does not mean that those compounds must be able also to inhibit CA polymerization and HIV-1 capsid assembly. To evaluate the inhibitory activity of GATG dendrimers on the polymerization of CA, we took advantage of the property of the purified recombinant CA (46 µM protein monomer concentration) to polymerize in vitro when incubated in the presence of high salt concentrations.48,59 Under these conditions, CA assembles into mainly tubular polymers with the same molecular organization as authentic mature HIV-1 capsids.5,6 Every [G1] dendrimer shown in Table 1 (except [G1]Lac) was assayed; as a negative control, [G2]-Lac, which did not bind to CTD, was also assayed. The results revealed that all but one of those dendrimers did not significantly inhibit CA polymerization, even at the highest dendrimer concentrations tested (up to 10-fold molar excess of dendrimer relative to CA). A representative result is shown using up to 460 µM [G1]-Man (a 10-fold molar excess over CA; Figure 6 A). In contrast, dendrimer [G1]-CO2Na was clearly able to inhibit CA polymerization (Figure 6B). The initial rate of the polymerization reaction of CA decreased from about 0.083 min-1 in the absence of dendrimer to about 0.028 min-1 in the presence of a 10-fold molar excess of [G1]-CO2Na. The amount of polymer formed was also reduced in these latter conditions. Experiments were also carried out in the presence of a macromolecular crowding agent to increase the chemical activity of the molecules involved.48 Under these conditions, the rate of CA assembly and the amount of polymer formed increased substantially, but the inhibition results with every dendrimer tested were similar to those obtained in the absence of macromolecular crowding conditions (data not shown).

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Figure 6. Analysis of the inhibitory activity of dendrimers on the assembly in vitro of the HIV-1 capsid protein. (A) Polymerization of CA in the conditions described in the Experimental Section, either in the absence (black line) or presence of the [G1]-Man dendrimer at a 5-fold molar excess (blue line) or a 10-fold molar excess (red line). (B) Polymerization of CA in the absence (black line) or presence of [G1]-CO2Na dendrimer at a 5-fold molar excess (blue line) or a 10-fold molar excess (red line).

Discussion First-Generation GATG Dendrimers Bind to the Dimerization Interface of CTD. In this work, we have tested the ability of a second generation of GATG dendrimers to bind CTD and to inhibit capsid assembly. Some dendrimers are able to specifically bind CTD at the residues that form helix 9 in the wild-type dimeric CTD. The first direct experimental evidence comes from the fluorescence studies, which show strong quenching in Trp184 of wild-type CTD upon binding of [G1]OSO3Na and [G1]-CO2Na (Figure 2). And the second evidence comes from the HSQC experiments, which allow us to map on a residue-by-residue basis the CTD binding region (Figure 5, Table 2). As it can be observed, all the residues shown to interact with the dendrimers are spatially close, being clustered around the position where the helix 9 should be formed (Figure 5), as well as at helices 10 and 11. Previous structural studies on wild-type dimeric CTD alone10,13 show that residues Thr148 and Ile150 (belonging to the 310 helix); Leu151, Asp152, and Arg154 (belonging to the MHR); Leu172, Glu175, Ala177, and Ser 178 (at helix 8); Glu180, Val181, Trp184, Met185, Glu1187, Thr188, Leu189, Val191, Gln192, and Asn193 (at the helix 9, the dimerization helix); Lys199 (helix 10); and Lys203 and Pro207 are involved in dimer-dimer contacts. Many of those residues are also energetically important for CTD dimerization,46 namely, Trp184, Met185, Leu189, Arg154, Leu151, Val181, Iel150, Leu172, and Glu175 (in decreasing order of energetic importance). Interestingly, several of the residues of the dimerization interface of CTD are also involved in the contact with the dendrimers, as judged by the changes in the intensities in HSQC experiments; specifically, residues Glu180, Trp184 (the mutated residue in the CTDW184A), Thr188, and Lys203 (Table 2, Figure 5). However, residues at the C terminus of the dimerization helix, such as Glu187, Thr188, Leu189, and Val191 (which are involved in the dimer-dimer interface), did not show any variation in the intensity of HSQC spectra. Moreover, Met185 and Leu189 are two of the three energetically most important residues (after Trp184) for CTD dimerization and they are not involved in binding of the GATG dendrimers. Thus, while the contact epitope in CTD for dendrimer binding does overlap with the contact and the energetic epitope for homodimerization, the overlap is limited. We think this might be the reason why all but one of the GATG dendrimers are not able to significantly hamper polymerization of CA.

The GATG dendrimers also appear to interact to a large extent with the MHR, although not all interact with the same residues (Table 2). A CTD mutant with Ala177 deleted69 is able to form a domain-swapped dimer, and the authors suggest that the MHRcontaining segment could be involved in the polymerization of Gag to form the immature capsid. This observation suggests that formation of the immature capsid could be inhibited by the dendrimers. The dendrimers also interact with the C-cap regions of the last two helices of CTD. These regions are involved in the binding to lysyl-tRNA synthetase, especially Thr210.70 Probably, the GATG dendrimers interact with those helices due to proximity at the C-terminal region of helix 9 and to their volume. However, it would be interesting to monitor whether the presence of the dendrimers is able to obstruct, to some extent, the interaction between CTD and the tRNA synthethase (which is in the order of nM). Finally, it could be thought that the binding of GATG dendrimers to those polypeptide regions induced large structural changes in CTD. Although we observed changes in the far-UV CD spectra of several dendrimers (Figure 3A), the fact that the HSQC spectra did not show any large changes in chemical shifts suggest that the tertiary structure is not significantly altered. Dominant Forces behind the Binding of GATG Dendrimers to CTD. The hydrophobic aromatic moieties of several CTDbinding peptides are involved in a large number of hydrophobic contacts with CTD, and it has been suggested that these contacts are the dominant driving force in peptide binding7,8,16,55,67,68 (see below). Likewise, hydrophobic contacts may also be energetically dominant in binding of the GATG dendrimers to CTD, because the dendrimers that caused intensity variations in a larger number of residues (Table 2) are those with a bulky side chain (Scheme 1), namely, [G1]-CO2Na and [G1]-SO3Na. However, hydrophobicity does not appear to be the only driving force in binding of the dendrimers; the fact that PEG[G1]-OSO3Na showed the largest changes in NMR signal intensities suggests that polar and hydrophilic interactions are also important, as it has been suggested for binding of other dendrimers to proteins.51,71 In fact, it has been suggested that the use of dendrimers to disrupt the tertiary and quaternary structures of proteins relies mainly on electrostatic interactions between protein and dendrimer as the driving force of the process.36,37,71,72


We have also found that the dimerization interface of CTD is not accessible to any [G2]-GATG dendrimer; in fact, only [G1] dendrimers are able to bind CTD, which suggests spatial restrictions to binding the dimer-dimer interface. There are other examples in the literature where dendrimer-protein interactions are also generation dependent.39,72 To sum up, our results suggest that (i) GATG-dendrimer binding does not seem to alter substantially the secondary and tertiary structures of the protein, as shown by the absence de changes in the HSQC spectra of CTDW184A; and, (ii) GATGdendrimer binding involves specific polypeptide patches of CTD (as shown above) in contrast to studies of other protein-dendrimer interactions that seem to be highly unspecific.51,63 Comparison of GATG Dendrimers with Other Molecules Able to Bind to CTD. There are several peptides that are able to bind to the CTD-CTD dimerization interface or to the NTD-CTD interface,7,8,16,55,67,68 which together with the NTD-NTD hexamerization interface, is important during viral assembly.6 The region of CTD involved in peptide binding is similar to the dimer-dimer interface described above: Ser149, Arg162, Val165, Asp166, Phe168, Tyr169, Leu172; Arg173, Leu151, Glu175, Gln176, Ala177, Ser178, Gln179, Glu180, Val181, Lys182, Asn183 Trp184, Met185, Thr186, Glu187, Thr188, Leu189, Leu190, Pro207, Ala 209, Thr210, Leu211, Met214, and Met215. The majority of the residues of CTD involved in peptide binding also appear to intervene in GATG-dendrimer binding (Table 2, residues in bold): Glu180, Lys182, Asn183, Ala184 (the mutation site), Thr186, Thr188, Leu190, Lys203, Thr210, and Cys218. Interestingly enough, the affinity of some of the GATG dendrimers for wildtype CTD is larger than the affinity of a peptide, which inhibits capsid assembly in vitro16 (∼15 µM), and larger than that of a peptide, which mimics the dimerization interface of CTD15 and also inhibit polymerization of CA in vitro (RB and MGM, unpublished results; ∼50 µM). We do not know yet the reasons why most dendrimers are not able to inhibit the polymerization of CA. As suggested above, they could be related to the fact that the dendrimer binding region only partially overlaps with the homodimerization interface of CTD, especially if the more restricted, energetic epitope, and not the whole contact epitope, is considered.46 It should be borne in mind that the dendrimers may bind the monomeric form of CTD, as analyzed here in the HSQC experiments, in a different way than when they bind the CTD dimer (see Results section), as the tertiary structure of the free monomer is rearranged upon monomermonomer association.45-47,50,66 We believe that, as indicated above, the dendrimer-binding features are similar in the monomeric and dimeric species. Furthermore, due to the broadening of most of the signals of residues involved in R-helix 9 in wild-type CTD, we were not able to map the dendrimer recognition binding region in the dimeric species (the broadening is due to the conformational exchange of the dimeric formation equilibrium14,70 and to the intrinsic high flexibility of the R-helix 947,66). However, it is important to note that [G1]-CO2Na did inhibit CA polymerization, which is what one could more easily expect from the CTD binding results. Our working hypothesis is that [G1]-CO2Na, but not the other [G1] dendrimers, may have the proper size and flexibility; furthermore, we suggest that [G1]-CO2Na can interact more easily than the other dendrimers with NTD, whose oligomerization NTD-NTD and NTD-CTD interfaces are important for capsid assembly.5,6 To test this latter hypothesis, we carried out far-UV CD experiments of the dendrimer-NTD complexes (data not shown); our results indicate that the [G1]-CO2Na modifies at a greater extent than the other dendrimers do the shape of the NTD far-UV CD spectrum, leading to a reduced ellipticity. Thus, [G1]-


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CO2Na interacts more strongly with NTD than the other organic molecules, and this interaction reduces the helical content of the domain, probably hampering capsid assembly during our experiments in vitro and, hence, polymerization of CA. Moreover, because the HSQC spectra of CTD did not change substantially in the presence of [G1]-CO2Na, we think that inhibition of capsid assembly by this dendrimer does not involve the presence of disordered species of CTD, but docking with CTD regions that intervene during CA assembly. Altogether, these findings indicate that [G1]-CO2Na can be considered as a new lead molecule for the design of novel anti HIV-1drugs targeting capsid assembly; for instance, by creating chimeras with functionalized peptides at each of the ends of the dendrimer or, alternatively, by changing the position and number of the branches in the dendrimer.

Conclusions We have shown, for the first time, that [G1]-GATG dendrimers are able to bind to the dimerization interface of CTD. One of these dendrimers hampers the assembly in vitro of the HIV-1 capsid and may provide a new lead compound for the development of new antiviral agents. Acknowledgment. We thank the reviewers for helpful suggestions and ideas. We thank Dr. Francisco N. Barrera for reading the manuscript, helpful discussion, and suggestions. This work was supported by FIPSE foundation (Exp: 36557/06) to J.L.N. and M.G.M., Spanish Ministerio de Ciencia e Innovacioń (SAF2008-05742-C02-01, CSD2008-00005 to J.L.N., SAF200407722 to A.V.-C., BIO2009-10092 to M.G.M., and CTQ200910963 to E.F.-M.), Generalitat Valenciana (ACOMP2010/114 to J.L.N.), Diputacioń General de Aragoń (PI078/08 and PI044/ 09 to A.V.C.), Xunta de Galicia (PGIDIT06PXIB209058PR to E.F.-M.), and an Institutional grant from Fundacioń Ramon Areces (to the CBMSO, where M.G.M. works). The stays of R.D. in the laboratory of A.V.C. were supported by the Spanish Ministerio de Ciencia e Innovacioń (BFU2008-02302-BMC). A.S.-H. thanks the Spanish Ministerio de Ciencia e Innovacioń for a FPU fellowship. We deeply thank May Garcıá, Marıá del Carmen Fuster, Raquel Jorquera, and Javier Casanova for technical assistance.

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