Anionic Carbosilane Dendrimers Destabilize the ... - ACS Publications

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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

Anionic Carbosilane Dendrimers Destabilize the GP120-CD4 Complex Blocking HIV‑1 Entry and Cell to Cell Fusion Carlos Guerrero-Beltran,†,●,‡,⬢ Ignacio Rodriguez-Izquierdo,†,●,⬢ Ma Jesus Serramia,†,● Ingrid Araya-Durán,§,□,∥ Valeria Márquez-Miranda,§,□,∥ Rafael Gomez,⊥,#,¶ Francisco Javier de la Mata,⊥,#,¶ Manuel Leal,■,▽ Fernando González-Nilo,§,□,∥ and M. Angeles Muñoz-Fernández*,†,●,‡,¶ †

Laboratorio InmunoBiología Molecular, Hospital General Universitario Gregorio Marañoń and Instituto de Investigación Sanitaria Gregorio Marañoń (IISGM), 28007 Madrid, Spain ● Spanish HIV HGM BioBank, 28009 Madrid, Spain ‡ Plataforma de Laboratorio, Hospital General Universitario Gregorio Marañoń , 28007 Madrid, Spain § Center for Bioinformatics and Integrative Biology (CBIB), Facultad de Ciencias Biológicas, Universidad Andres Bello, Av. República 239, Santiago, Chile, 8370146 □ Fundación Fraunhofer Chile Research, Las Condes, Chile, 7550296 ∥ Centro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, Valparaíso, Chile, 2360102 ⊥ Department of Química Orgánica y Química Inorgánica and #Instituto de Investigación Química “Andrés M. del Río″ (IQAR), Universidad de Alcalá (IRYCIS), Campus Universitario, 28871 Madrid, Spain ¶ Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III, Av. de Monforte de Lemos, 5, 28029 Madrid, Spain ■ Instituto de Biomedicina de Sevilla (IBiS). Hospital Universitario Virgen del Rocio, Av. Manuel Siurot, s/n, 41013 Sevilla, Spain ▽ Servicio de Medicina Interna. Hospital Viamed Santa Á ngela, Av. de Jerez, 59, 41014 Sevilla, Spain ABSTRACT: Cell-to-cell transmission is the most effective pathway for the spread of human immunodeficiency virus (HIV-1). Infected cells expose virus-encoded fusion proteins on their surface as a consequence of HIV-1 replicative cycle that interacts with noninfected cells through CD4 receptor and CXCR4 coreceptor leading to the formation of giant multinucleated cells known as syncytia. Our group previously described the potent activity of dendrimers against CCR5tropic viruses. Nevertheless, the study of G1-S4, G2-S16, and G3-S16 dendrimers in the context of X4-HIV-1 tropic cell−cell fusion referred to syncytium formation remains still unknown. These dendrimers showed a suitable biocompatibility in all cell lines studied and our results demonstrated that anionic carbosilane dendrimers G1-S4, G2-S16, and G3-S16 significantly inhibit the X4-HIV-1 infection, as well as syncytia formation, in a dose dependent manner. We also demonstrated that G2-S16 and G1-S4 significantly reduced syncytia formation in HIV-1 Env-mediated cell-to-cell fusion model. Molecular modeling and in silico models showed that G2-S16 dendrimer interfered with gp120-CD4 complex and demonstrated its potential use for a treatment.

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INTRODUCTION

two-thirds of infected patients; however, in regions like Eastern Europe and Central Asia, those data do not reach 30%,1 so they are more susceptible to CXCR4-tropic viruses and developing AIDS. The envelope glycoproteins (Env) of HIV-1 are the key elements involved in viral entry. Env derives from gp160 precursors generating noncovalently associated trimers of gp120 and gp41 heterodimers. Thus, the entry of HIV-1 into susceptible cells begins with the initial binding of the HIV-1

Human immunodeficiency virus type 1 (HIV-1) continues to be a major global public health problem. The UNAIDS program on HIV/AIDS estimated that 36.7 million people lived with HIV-1 worldwide, highlighting that around one-third of all people did not know their HIV-positive status.1 The percentage of anti-retroviral naïve individuals harboring X4 viruses varies between 10% and 38%. Some studies estimated the prevalence of CXCR4-using virus in primary HIV infection at 6−19%.2 Therefore, it is important encourage treatment during this phase of infection. Otherwise, the anti-retroviral therapy coverage according to data from UNAIDS is increasing in over © XXXX American Chemical Society

Received: February 12, 2018 Revised: March 23, 2018 Published: March 23, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00106 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

Figure 1. Viability studies of carbosilane dendrimers. MTT assays were performed on (a) MT2, (b) HeLa P5, (c) HeLa ADA, and (d) HeLa 243 cell lines. These cell lines were treated with increased concentrations range (0.1−20 μM) of G1-S4, G2-S16, and G3-S16 dendrimers. Nontreated (NT) cells were used as cell viability control, DMSO was used as cell death control. Data were represented as mean ± SD of three individual experiments performed in triplicate. Abbreviations: NT = untreated control; DMSO = Dimethyl sulfoxide.

In this sense, nanotechnology has emerged as a new field to prevent several infectious diseases in recent decades. In particular, polyanionic carbosilane dendrimers are potential candidates against sexually transmitted viruses due to their unique characteristics.12,13 Our group has widely studied the G1-S4, G2-S16, and G3-S16 carbosilane dendrimers with sulfonate G2-S16 or sulfate groups G1-S4 and G3-S16 at the periphery, in the HIV-1 infection context.12,14 In this work, we study these dendrimers to prevent X4-HIV-1 infection and to impede cellto-cell X4-HIV-1 tropic virus infection.

Env, gp120, with the host CD4+ T cell membrane receptor (CD4-gp120 complex) and induces conformational changes in Env, both to expose epitopes for subsequent interaction with CCR5 or CXCR4 coreceptors and to activate the gp41 transmembrane subunits for membrane fusion.3 CCR5 is expressed in conjunction with the CD4 surface mainly on activated lymphocytes, macrophages, dendritic cells, and neuronal cells, whereas CXCR4 with CD4 is found on the surface of monocytes and resting T cells.4 In agreement with the coreceptors used, HIV-1 strains are subdivided into CCR5-(R5) and CXCR4-(X4) tropic viruses. Although CCR5 and CXCR4tropic viruses can be isolated from body fluids, CCR5-tropic viruses are exclusively associated with primary HIV-1 infection, with the asymptomatic phase of AIDS, which coincides with acute infection.5 The switch from R5 to X4 viruses is associated with the loss of CD4+ T cells and AIDS development, with the symptomatic phase of AIDS.6−8 The HIV-1 coreceptor ratio influences cell susceptibility to infection and contributes to viral pathogenesis. Subsequently, nuclear fusion occurs within the CXCR4-tropic viruses, causing syncytia formation and apoptosis.9 Although HIV-1-induced T cell-based CXCR4-tropic viruses have been considered short-lived artifacts of in vitro cell culture, it has been shown that when cultivated in 3D hydrogels, which more closely resemble the environment in lymphoid tissue of HIV-1+ individuals, virus CD4+ T cells can form small syncytia that closely mimic those observed in lymph nodes of humanized mice and possibly in secondary lymphoid tissue of HIV-1 infected individuals.10 Using a 3D in vitro system proves that such small T cell-based syncytia can transfer virus to uninfected cells.11 Thus, given that cell-to-cell transmission provides a means of evading anti-retroviral therapies and antibodymediated responses, it is important to emphasize that preventive and therapeutic approaches should take into consideration the unique aspects of this mode of infection.

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RESULTS AND DISCUSSION Biocompatibility of Polyanionic Carbosilane Dendrimers. Our group has widely studied carbosilane dendrimers as potential molecules to prevent viral infections. We previously described the potent activity of carbosilane dendrimers against sexually transmitted viruses such as HIV-1 and herpes simplex virus type 2 (HSV-2).13,15−18 To achieve a major understanding about the underlying mechanism to this inhibition, we studied the potential activity of three selected dendrimers, G1-S4, G2-S16, and G3-S16, to inhibit X4-HIV-1NL4.3 strain, associated with the symptomatic phase of AIDS. The X4-HIV1NL4.3 infection was quantified as syncytia formation, and we focused on the mechanism of action, supported with in silico models. Cell viability of our three polyanionic carbosilane dendrimers, with sulfonate G2-S16 or sulfate groups G1-S4 and G3-S16 at the periphery, with a nanoscopic size, versatility, and multibranching properties was studied on MT2, HeLa P5, HeLa ADA, and HeLa 243 cell lines by MTT assay. These cell lines were seeded and treated with G1-S4, G2-S16, or G3-S16 dendrimer in a range of concentration from 0.1 μM to 20 μM to determine the maximum nontoxic concentration (Figure 1a,b,c,d). Dendrimers were considered nontoxic when the survival rate B


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Figure 2. Anti-X4-HIV-1 activity of dendrimers quantified as syncytia formation. (a) Representative confocal images and (b) counting of syncytia/ confocal field. MT2 cells were treated with G1-S4, G2-S16, or G3-S16 dendrimer at different concentrations and then infected with X4-HIV-1NL4.3 for 2 h. After 72 h, cells were immune-stained for CD4 and DAPI. TDF (1 μM), T20 (20 μM), and SAQ (5 μM) were used as antiviral controls. Data represent the mean ± SD of three independent experiments performed in triplicate. ***: p < 0.0001 vs CI. Abbreviations: CI = control of infection, TDF = Tenofovir Disoproxil Fumarate, SAQ = Saquinavir.

quantification of syncytia formation, p24gag enzyme-linked assay, and titration on TZM.bl cell line. Overall, the G1-S4, G2-S16, and G3-S16 dendrimers inhibited the X4-HIV-1NL4.3 infection in a dose response manner (Figure 2a,b). G1-S4 and G2-S16 inhibited X4-HIV1NL4.3 infection in a significant manner from 0.5 μM; indeed, a significant reduction of syncytia formation was observed at 5 μM, 10 μM, and 15 μM for both dendrimers. However, G3-S16 dendrimer prevented the infection from 0.1 μM at 1 μM. Confocal imaging revealed a reduction in the number and size of syncytia, mainly for G1-S4 and G2-S16 dendrimers compared to X4-HIV-1NL4.3 control of infection (Figure 2a). These data are related with the quantification of syncytia formation by random counting of ten confocal fields (Figure 2b). Based on this data, we further assayed the presence of X4-HIV-1NL4.3 in the supernatants, as well as its infectivity. The p24gag enzyme-linked assay showed a significant reduction of X4-HIV-1NL4.3 quantity for G1-S4 and G2-S16 dendrimers at all tested concentrations. A decrease >90% was observed at 5 μM for G1-S4 and at 1 μM for G2-S16 dendrimer. G3-S16 dendrimer reached a significant reduction

was >80%. Nontreated (NT) cells were used as cell viability control and dimetil sulfoxide (DMSO) 10% was used as death cellular control. G1-S4 dendrimer was nontoxic up to 20 μM in MT2, HeLa P5, and HeLa ADA cell lines, and up to 10 μM in HeLa 243 cell line. G2-S16 was nontoxic at maximum concentration assayed (20 μM) in all cell lines tested. Nevertheless, G3-S16 maximum nontoxic concentration was 1 μM in all cell lines. Thus, G3-S16 was the dendrimer with highest toxicity rates due to its major generation and higher size. Moreover, G1-S4 and G3-S16 dendrimers with sulfate groups in the surface were more toxic than G2-S16 sulfonate dendrimer, showing that not only are the generation and size determinants for the biocompatibility, but also the functionalizing molecules. Dendrimers Prevent X4-HIV-1NL4.3 Infection. Once the biocompatibity of the dendrimers was demonstrated, we evaluated the capability of the three dendrimers to inhibit the X4-HIV-1 infection. Thus, MT2 cells were treated with a concentration range up to maximum nontoxic concentration for 1 h and infected with X4-HIV-1NL4.3 strain. The X4-HIV1NL4.3 infection was evaluated by confocal microscopy as the C


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Figure 3. Quantification and infectivity of X4-HIV-1NL4.3. (a) HIV-1 quantity measured as p24gag enzyme-linked assay following manufactureŕs instruction and (b) infectivity of viral particles by titriation on TZM.bl cells. Previously seeded cells were treated with supernatants for 2 h. HIV-1 infection was measured as luciferase activity at 48 h post-infection. Data represent the mean ± SD of three independent experiments performed in triplicate *: p < 0.05, **: p < 0.001, ***: p < 0.0001 vs CI. Abbreviations: CI = control of infection.

syncytia formation at 0.5 μM, although we observed a 3-fold reduction of multinucleated cells at 1 μM (Figure 4a,b). The p24gag enzyme-linked assay revealed a significant decrease in the quantity of X4-HIV-1NL4.3 for G1-S4 and G2-S16 dendrimers at 5 μM and 10 μM concentrations. In the case of G3-S16 dendrimer, the reduction was significant only at 1 μM (Figure 5a). These results are in agreement with those obtained by counting confocal fields (Figure 5a). We also observed a significant decrease in the infectivity of the X4-HIV-1NL4.3 strain in the supernatants obtained from G1-S4, G2-S16, and G3-S16. Although p24gag enzyme-linked showed a decrease about 50% in the quantity of viral particles for G3-S16, supernatant titration for this dendrimer at 0.5 μM and 1 μM demonstrates a significant decrease in the infectivity of those viral particles, similar to those reached for G1-S4 at 5 μM (Figure 5b). Data revealed that once the MT2 cells were infected with X4-HIV-1NL4.3 strain and treated with G1-S4 and G2-S16 dendrimers, inhibition of syncytia formation was observed at 5 μM and 10 μM (maximum inhibitory concentrations) and reduced the quantity of viral X4-HIV-1NL4.3 particles and their infectivity. G3-S16 did not prevent completely the syncytia formation at any concentration tested, although the differences were significant (3-fold reduction of multinucleated cells at 1 μM). All these results observed by confocal microscopy were strongly supported by the supernatant titration by p24gag enzymelinked assay, as well as titration on TZM.bl cells. Results revealed a significant decrease in viral production mediated by G1-S4 and G2-S16 dendrimers compared with control of HIV-1 infection and subsequent loss of viral infectivity. These data demonstrated that both dendrimers not only prevented X4-HIV-1 infection but also played an important role in induced HIV-1 cell fusion. In this sense, the nanotechnology not only prevented new HIV-1 infections13,16,17,21,22 but is also crucial in the development of novelty treatments for HIV-1 infected individuals. It is important to keep in mind the number of HIV-1 infected individuals and that every day there are new HIV-1 infections in the world. These new HIV-1 infections induce a general deregulation of the immune system where the majority of immune cells lose their functional ability. T and B cell exhaustion is characterized by the increase of an activated phenotype, decrease of proliferative ability, and loss of their effector capacity. These outcomes are related to uncontrolled viral

of >75% at 0.5 μM and >95% at 1 μM also in a dose-dependent manner (Figure 3a). The reduction of viral particles is in accordance with the reduction of infectivity previously observed in confocal microscopy. Inhibitions >90% were obtained for G2-S16 at all concentrations tested. Nevertheless, G1-S4 dendrimer obtained this percentage from 5 μM to 15 μM and G3-S16 reached the 95% of inhibition at 1 μM, the highest concentration tested (Figure 3b). These results exerted a potent anti-X4-HIV-1NL4.3 activity for all dendrimers due to at least one concentration reached inhibition rates >90%. Data demonstrated the anti-X4-HIV-1NL4.3 activity of G1-S4, G2-S16, and G3-S16, and the dose−response action. G1-S4 and G2-S16 reached their maximum inhibition rate >99% at 5 μM. In the case of G3-S16, values nearby 95% of inhibition against X4-HIV-1NL4.3 strain were reached at 1 μM (maximum nontoxic concentration). Our group already demonstrated that those dendrimers blocks the infection acting as entry inhibitors;12,14 thus, we hypothesize the same behavior in the case of CXCR4-tropic viruses. Dendrimers Reduce the Syncytia Formation in MT2 Infected Cells. We further studied whether G1-S4, G2-S16, and G3-S16 dendrimers inhibited syncytia formation once the cells were infected with X4-HIV-1NL4.3 strain. Viral cell-to-cell transmission represents the most effective pathway transmission due to the direct contact between infected and noninfected cells, which increase infection rate. Env gp120 subunit interacts with CD4 receptor and a coreceptor, typically CXCR4, on the surface of target cells, resulting in multinucleated cells known as syncytia. Those syncytia were typically related with in vitro artifact. However, they had been observed in lymph nodes of HIV-1-infected humanized mice and individuals virological phenotype switches under salvage therapy with lopinavir-ritonavir in heavily pretreated HIV-1 vertically infected children.19,20 To test that mechanism, MT2 cells were infected, as previously described in M&M 4.5, and treated with G1-S4, G2-S16, or G3-S16 dendrimer at their maximum inhibitory concentrations at 48 h post-infection. Confocal images showed that G1-S4, G2-S16, and G3-S16 dendrimers reduced syncytia formation in MT2 cells infected previously by X4-HIV-1NL4.3 strain (Figure 4a,b). The counting of syncytia per confocal microscopy field exerted that G1-S4 reduced 3-fold syncytia formation at 5 μM, and even more at 10 μM. G2-S16 dendrimer almost completely diminished syncytia formation at both 5 μM and 10 μM. G3-S16 did not reduce D


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Figure 4. Inhibition of syncytia formation by dendrimers. (a) Representative confocal images and (b) counting of syncytia/confocal field. MT2 cells were infected by X4-HIV-1NL4.3 for 2 h and treated with G1-S2, G2-S16, or G3-S16 dendrimer at 48 h post-infection. After 72 h, cells were immunestained for CD4 and DAPI. Data represent the mean ± SD of three independent experiments performed in triplicate. ***: p < 0.0001 vs CI. Abbreviations: CI = control of infection.

Figure 5. Supernatant titration. (a) X4-HIV-1NL4.3 quantity measured as p24gag enzyme-linked assay by following manufactureŕs instruction and (b) infectivity of viral particles by titration on TZM.bl cells. Previously seeded cells were treated with MT2 infected cell supernatants for 2 h. HIV-1 infection was measured as luciferase activity at 48 h post-infection. Data are represented as the mean ± SD of three independent experiments performed in triplicate. **: p < 0.001, ***: p < 0.0001 vs CI. Abbreviations: CI = control of infection.

persistence and disease progression.23,24 New approaches, such as the use of dendrimers to prevent syncytia formation in the absence of anti-retroviral therapy or their use in combination with some anti-retrovirals could play an important role in the control not only of R5-HIV-1 but also of X4-HIV-1. HIV-1 Env-Mediated Cell-to-Cell Fusion Assay. We further designed an in vitro cell-to-cell fusion model to assess whether our dendrimers were acting directly on the fusion process. That model consists of a coculture of HeLa P5 cell line

(stably transfected with human CD4 and CCR5- GFP cDNA and an HIV-LTR-driven-gal reporter gene) with HeLa 243 or HeLa ADA cells, that co-expresses Tat and Env HIV-1 X4- and CCR5-tropic, respectively. In that sense, HeLa P5 cell line acts masking HIV-1, and HeLa 243 or HeLa ADA cells function as cellular receptors and coreceptors, respectively. When gp120gp41-mediated fusion occurs, Tat protein trans-activates LTR promoter at HeLa P5 nuclei, resulting in syncytia formation and β-galactosidase expression. E


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Figure 6. HIV-1 Env-mediated cell-to-cell fusion model. HeLa P5 cells were coculture at ratio 1:1 with HeLa ADA or HeLa 243 cell lines. After 16 h, syncytia formation was quantified by β-galactosidase activity for (a) HeLa P5-ADA and (b) HeLa P5-243 coculture. β-Galactosidase substrate (X-Gal) was used to obtain representative images of (c) HeLa P5-ADA and (d) HeLa P5-243 cocultures. T20 (20 μM) was used as inhibitor control. Data were represented as mean ± SD of three individual experiments performed in triplicate. *: p < 0.05, **: p < 0.001, ***: p < 0.0001vs NT. Abbreviations: NT = nontreated coculture. T20 = Enfuvirtide.

HeLa P5 cocultures with HeLa ADA or HeLa 243 were treated with a concentration of dendrimers with previously observed inhibitory activity (G1-S4:10 μM, G2-S16:10 μM, and G3-S16:1 μM). Cell culture and coculture were used as nonfusion control or fusion control (NT), respectively, and T-20 was used as fusion inhibitor. Unexpectedly, in HeLa P5 and HeLa ADA coculture, β-galactosidase expression for G1-S4 dendrimer was similar to nontreated coculture (NT) (Figure 6a). However, G1-S4 inhibited HeLa P5 and HeLa 243 cellular fusion (Figure 6b). On the other hand, G2-S16 significantly reduced the cellular fusion in both HeLa P5 cells merging with HeLa 243 cells and HeLa ADA cells (Figure 6a,b). G3-S16 did not prevent cellular fusion neither in HeLa P5−HeLa ADA nor in HeLa P5−HeLa 243 cells (Figure 7a,b). These results were confirmed by X-Gal images, where G2-S16 dendrimer destabilized the interaction between gp120-CD4 (Figure 6c,d). Thus, this model showed that dendrimer G1-S4 blocked the P5-243 cell fusion, but not the P5-ADA cells, indicating a specific and powerful mechanism of action against gp120-CD4 CXCR4 coreceptors. Nevertheless, G2-S16 dendrimer blocked the cellular fusion in all cases, with a behavior like enfuvirtide, which is an entry inhibitor of HIV-1 infection. Probably, G2-S16 dendrimer was affecting to the gp120-CD4 interaction, which means that G2-S16 is a potent and versatile entry inhibitor. Regarding the G3-S16 dendrimer, no significant inhibitory values on this cellular fusion model were obtained. Summarizing, we demonstrated by cell-to-cell fusion model that G2-S16 dendrimer interferes with gp120-CD4 interactions. Those data suggest that G2-S16 dendrimer could be used in a combined therapy with other anti-retrovirals, increasing their efficacy, and moreover, including another target since it does not exist in any

powerful combination with entry inhibitors, providing the possibility to diminish the virological failure. Given the low anti-HIV activity and low genetic barrier for resistance to enfuvirtide, the only anti-retroviral inhibitor of viral entry currently used in therapy,25 G2-S16 rises as a promising candidate, based on its high activity against both CCR5 and CXCR4-tropic viruses. G2-S16 Dendrimer Affects gp120-CD4 Interactions. In order to confirm the G2-S16 mechanism of inhibition, we evaluated the gp120-CD4 interactions with G2-S16 dendrimer by molecular modeling in X4-HIV-1 (Figure 7a) and R5-HIV-1 (Figure 7a) interactions. The evolution of Root Means Square Distance (RMSD) of the CD4 contact zone along molecular dynamics (MD) simulation (Figure 7c) indicated that, when in contact with X4-gp120, more fluctuations were observed in RMSD profile than those observed with R5-gp120. This effect may be attributed to the G2-S16 dendrimer, which could be inducing more perturbation in X4-gp120-CD4 than in R5-gp120-CD4 complex. To confirm these results, the solvent accessible surface area (SASA) of the CD4 contact zone was evaluated (Figure 7d). The data showed higher values when it is interacting with X4-gp120 compared to when it is interacting with R5-gp120. Once again, this may represent that G2-S16 dendrimer binding alters the conformation of the complex to a higher extent when X4-gp120 is involved. To explain the results discussed above, and to verify if those changes are occurring due to G2-S16 dendrimer interaction with either gp120 or CD4, intermolecular contacts between dendrimer and gp120 or CD4 were calculated considering those atoms of dendrimer falling within 3.5 Å of any atom in gp120 or CD4. G2-S16 dendrimer tends to interact in a stable way with R5-gp120 during the entire simulation time (Figure 7e). In contrast, G2-S16 dendrimer establishes few contacts with F


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X4-gp120 at the beginning, but they increase progressively over the simulation time until they reach similar values to those calculated with R5-gp120. Meanwhile, G2-S16 dendrimer showed that it established only a few contacts with CD4 when it was in complex with R5-gp120 (Figure 7f). We verified a progressive increase in the number of contacts between CD4 and the G2-S16 dendrimer during the simulation. This difference in the interaction with CD4 between the two gp120 variants could explain the higher destabilization observed in X4 variant in RMSD analysis. Figure 7g,h shows the location of the dendrimer in the complex X4-gp120-CD4, indicating those contact points that are being lost after G2-S16 dendrimer penetration in the gp120-CD4 contact zone. Those carbon contact points include residues VAL 421 and PRO 123 from gp120, and ASP 63 and SER 60 from CD4. To verify whether the binding between CD4 and gp120 is effectively disrupted due to the presence of the G2-S16 dendrimer, causing a decrease in the affinity between both proteins, the binding energy of gp120-CD4 complexes, in the presence and in the absence of the G2-S16 dendrimer, was calculated for each one of the gp120 variants R5 or X4. Thus, we have observed that, even when X4-gp120-CD4 complex displays a higher affinity than for R5 variant (−79.4 vs 68.2 kcal/mol, respectively), this binding energy value has a greater effect on dendrimer binding (−74.4 kcal/mol for X4 and −65 kcal/mol for R5). This behavior was in accordance with those checked with RMSD and SASA calculations of the CD4 contact zone, whereas contact points were also calculated, demonstrating that dendrimer exerts more influence on the perturbation of X4-gp120-CD4 complexes. As Table 1 depicts, ΔΔGBINDING, e.g., the ΔG difference between bound and unbound G2-S16 dendrimer states, is about +5 kcal/mol for X4 and only +3.2 kcal/mol for R5 variant, where positive values mean that in both systems gp120 and CD4 lose affinity upon interacting with the G2-S16 dendrimer. To sum up, as has been stated by Nandy et al. on SPL7013 dendrimer,26 G2-S16 dendrimer does not cause the formation of gp120-CD4 complexes in correct numbers. As a consequence, it prevents HIV-1 entry into the cells. This effect is more evident in the case of X4-HIV-1 than the R5-HIV-1 strain. Even though the G2-S16 dendrimer does not appear to directly block CD4 binding site of gp120, the contact zone between both proteins showed perturbations, as demonstrated by RMSD, by a number of contact points and by SASA analyses. Then, binding energy calculations also showed that, upon G2-S16 dendrimer binding to the complex, some interactions between gp120 and CD4 are lost. It means that the energetic differences between the dendrimerbound and dendrimer-unbound states are higher when HIV-1 X4-gp120 is involved. This may imply that dendrimer causes a more noticeable effect in HIV-1 X4-gp120-CD4 complexes. Our experimental results on G2-S16 dendrimer showed a significant decrease in the stability of X4-HIV-1 to bind to target cells in a more efficient manner than R5-HIV-1. This may be due to a perturbation in the ability of gp120 proteins from HIV-1 envelope to interact with CD4 receptors, as shown by molecular dynamics results. Thus, G2-S16 could impart both X4-gp120-CD4 and R5-gp120-CD4, demonstrating that could be used as entry inhibitor in an effective therapy against in HIV-1, and confirming the potential of G2-S16 for further studies.

Figure 7. Snapshots of the top ranked Patchdock/Firedock structures showing the binding position of the G2-S16 dendrimer with the best energy score. Gp120 is shown in green, CD4 in purple, and the G2-S16 dendrimer in cyan in (a) X4-HIV-1 or (b) R5-HIV-1. (c) Evolution of root-mean-square distance (RMSD) over simulation time, obtained from contact zone between CD4 and gp120 protein. (d) Time evolution of solvent accessible surface area (SASA), considering contact zone between CD4 and gp120 protein. (E) Variation of the number of contact points between dendrimer and Gp120 (any contact within 3.5 Å) during the simulation. (F) Variation of the number of contact points between dendrimer and CD4 over the simulation, considering systems where CD4 is interacting with either R5 or X4 gp120 variants (any contact within 3.5 Å). (G) Snapshot of the dendrimer/CD4/X4-gp120 complex obtained after 100 ns of time simulation, where gp120 is depicted in green, CD4 in purple, and the G2-S16 dendrimer in orange, whereas solvent accessible surface area of the contact zone of CD4 is depicted in yellow. (H) The X4-gp120/CD4 interface in the presence of the dendrimer. Image depicts contact points that are being lost after dendrimer penetration in the CD4/gp120 contact zone. Green line in plots represent CD4 and R5 gp120 variant, whereas blue line depicts CD4 and X4 gp120 variant.

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EXPERIMENTAL PROCEDURES Cell Culture and Viral Strains. TZM-bl cell line (ATCC, Manassas, USA), epithelial HeLa-derived cell line; HEK-293T G


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Results were obtained from last 20 ns of each MD simulation. a

ΔΔGBINDING (bound dendrimer/unbound dendrimer)

+5.06 +3.2 −74.4 −65 −8732.4 ± 23.6 −4797.6 ± 6.8 −2501.7 ± 9.1 −2512.4 ± 3.7

(gp120/CD4)

cell line (ATCC HTB-112, Manassas, USA), epithelial cell derived from a human endometrial carcinoma; HeLa P5 cell line, stably transfected with human CD4 cells, CCR5-GFP cDNA and an HIV-LTR-driven-gal reporter gene; HeLa 243 and HeLa ADA cell lines coexpressed Tat and Env HIV-1 CXCR4-tropic and CCR5-tropic, respectively. All were grown as previously described.27,28 MT2 cells (ATCC, Manassas, USA), HTVL-1 transformed human T-cell leukemia cells were cultivated in RPMI 1640 medium (Gibco, UK) supplemented with 10% FBS. Viral stocks of X4-HIV-1NL4−3 laboratory strain were obtained by transient transfection of pNL4−3 plasmid (NIH AIDS Research and Reference Reagent Program) in HEK-293T cell line. Stocks were clarified prior to evaluatioin of viral titer by HIV-1 p24gag enzyme-linked immunosorbent assay kit (INNOTEST; Innogenetics, Ghent, Belgium). Dendrimers and Reagents. Anionic carbosilane dendrimers G1-S4 and G3-S16 with 4 or 16 sulfate groups in the periphery, respectively, and G2-S16 with 16 sulfonate groups in the periphery were synthesized and tested according to methods reported by the Dendrimers for Biomedical Applications Group of University of Alcalá (Madrid, Spain).29,30 Stock Solution of dendrons (10 mM) and subsequent dilutions to working concentrations were prepared in nuclease-free water (Promega, Madrid, Spain). The schematic structures of the polyanionic carbosilane dendrimers are represented in Figure 8. Reagents used as controls were Tenofovir Disoproxil Fumarate (TDF; SelleckChem, Houston, TX, USA), enfuvirtide (T-20; Genentech, South San Francisco, CA, USA), and Saquinavir (SAQ; Invirase, Roche, Basel, Switzerland). Cell Viability Assays. The 3-(4-5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Sigma, St Louis, USA) was used according to manufacturer’s instruction in order to determine the viability of dendrimers on MT2, HeLa P5, HeLa 243, and HeLa ADA cell lines. Briefly, cells were treated with various concentrations of dendrimers (0.1−20 μM) and 24 h for MT2 cells or 72 h post-treatment in HeLa cells, MTT was added to the cultured cells. After 2 h, the reaction was stopped by adding DMSO, and the sample was evaluated using a plate reader. DMSO was used as death cellular control. Anti-X4-HIV-1 Activity of Dendrimers. MT-2 cells were seeded in eppendorfs (75 × 104 cells/eppendorf) and were pretreated with G1-S4, G2-S16, and G3-S16 dendrimers or TDF, T-20 and SAQ controls for 1 h at 37 °C before infect with X4-HIV-1NL4.3 strain (15 ng p24/106 cells). After 2 h, cells were centrifuged at 1500 rpm for 10 min, washed twice, and seeded in 12-well plates. At 72 h post-infection, syncytia formation was observed and samples were immune-stained for confocal imaging studies (M&M 2.6). Inhibition of X4-HIV-1 Syncytia Formation. MT2 cells were infected with X4-HIV-1NL4.3 strain (15 ng p24/106 cells), and 48 h post-infection, MT2 cells were treated with G1-S4, G2-S16, and G3-S16 dendrimers. Syncytia formation was observed at 72 h post-infection either by confocal microscopy or by supernatant titration. Immunofluorescence and Confocal Images. Microscope slides were treated with Poli-L-Lysine (Sigma-Aldrich, St. Louis, MO, USA) for 2 h. Slides were washed twice and MT2 cells were adhered. Cells were fixed with 4% paraformaldehide (PFA; Panreac, Barcelona, Spain) and immune-stained for CD4+ T cells (Immunotech, Marseille, France) for 1 h. After that, MT2 cells were washed and treated with 4′,6-diamidino-2phenylindole (DAPI) for nucleus observation. MT2 cells were visualized by using a Leica TSC SPE confocal microscope

−6156.3 ± 25.6 −7375.0 ± 8.87 −79.4 −68.2 −2503.8 ± 6.5 −2509.2 ± 3.7 X4 R5

−8619.4 ± 13.7 −7356.2 ± 6.9

GTOTAL (gp120) GTOTAL (CD4)

−6043.6 ± 11.0 −4778.7 ± 5.2

BINDING (bound dendrimer)

ΔG GTOTAL (gp120) GTOTAL (CD4) GTOTAL (gp120/CD4 complex) ΔGBINDING(unbound dendrimer) (gp120/CD4) GTOTAL (gp120/CD4 complex) gp120 variant

Table 1. Energetic Components, Relative Binding Energies, and ΔΔGBINDING (kcal/mol) Obtained by MM-GBSA Method for Each Gp120/CD4 System, Considering X4 and R5 Variantsa


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Figure 8. Molecular representation of dendrimers. (a) G1-S4 with 4 sulfate end groups, (b) G2-S16 with 16 sulfonate end groups, and (c) G3-S16 with 16 sulfate end groups. The generation of dendrimers is determined by considering that each generation corresponds to the number of repeating layers of silicon atoms.

G2-S16 Dendrimer−Protein Docking Simulations. Following the methodology proposed by Nandy et al.,26 where it was verified that SPL7013 dendrimer can destabilize the HIV-1 gp120−CD4 complex, a dendrimer−protein docking was carried out using the PATCHDOCK server (http://bioinfo3d.cs.tau.ac. il/PatchDock). PATCHDOCK is a freely available web server for molecular docking of protein−protein and protein−small molecule complexes, mainly supported by a geometry-based algorithm, which searches for docking transformations that yield good molecular shape complementarity. This server does not have limits for the number of atoms of ligand molecules. Complexes predicted by this server are later rescored using Firedock,38 which provides a refinement of the obtained structures. Best-scored protein−dendrimer complexes were then used to build molecular systems for molecular dynamics simulations. Molecular Systems. To evaluate how G2-S16 dendrimer exerts an effect on the gp120-CD4 interaction, two molecular systems were built, considering either R5-gp120 or X4-gp120 proteins, which were in complex with CD4 and G2-S16 dendrimer (bound-dendrimer state), considering the best-scored conformations obtained from protein−dendrimer docking calculations. Two additional systems, each one considering R5 or X4-gp120 in complex with CD4, but without dendrimer (unbound-dendrimer state), were built under the same conditions as the previous ones, for comparison. Thus, four systems were considered in this study. VMD software was used for visualization and building systems. Complexes were placed into a TIP3P39 water box, 124 × 119 × 138 Å3, with 150 mM of NaCl. Systems were subjected to a conjugate gradient minimization run. Later, NPT molecular dynamics simulations were carried out, using NAMD software package40 by about 100 ns each. Periodic boundary conditions were taken into account, and also the Langevin dynamics scheme to keep the temperature constant at 310 K, with a damping coefficient 1 ps−1. Langevin piston procedure41 was employed to maintain the pressure at 1 atm. Particle-mesh Ewald (PME) method42 for long-range electrostatic interactions was used with a real space cutoff of 9 Å. Van der Waals interactions were smoothly switched off at 10−12 Å. The RATTLE algorithm43 was used to constrain the covalent bonds between heavy and hydrogen atoms to the equilibrium length. To integrate equations of motion, the Verlet r-RESPA algorithm44 was used, with a time step of 2 fs. Energetic analyses for each molecular system were performed considering 200 gp120-CD4 snapshots from systems without dendrimer, taken from the equilibrated phase of the last 20 ns of MD trajectory. The same procedure was carried out for

(Leica Microsystems, Wetzlar, Germany). Fluorescence intensity was analyzed using ImageJ software (National Institute of Health, USA). Supernatant Tritiation. To determine the infectivity, ELISA HIVp24gag (Innogenetics, Ghant, Belgium) was used by the following manufacturer’s instruction. TZM.bl cells were seeded in 96-well plates (15 × 103 cell/well). After 24 h, medium was removed and replaced with 50 μL of supernatants for 2 h. TZM.bl cells were washed twice and medium was added. Forty-eight hours later, TZM.bl cells were lysed and luciferase activity was measured with BioTek Synergy 4 Hybrid Microplate Reader. HIV-1 Env-Mediated Cell-to-Cell Fusion Assay. HeLa 243 or HeLa ADA cell lines were coincubated with HeLa P5 cell line in 96-well plates at a 1:1 ratio for 16 h, as previously described.27 For a quantitative measurement of syncytia formation, galactosidase activity was evaluated by chemiluminiscence, using the kit β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer (Promega Corporation, Fitchburg, WI, USA). Galactosidase substrate 5-bromo-4-chloro-3-indoyl-Dgalactopyranoside (X-Gal) was used to show an overview of syncytia formed. Homology Modeling. Following the methodology reported in previous articles,31,32 R5-Gp120 sequence having a V3 loop was downloaded from HIV-1 database in Los Alamos National Laboratory (http://www.hiv.lanl.gov/) (Los Alamos ID: AY426111). X4-Gp120 sequence was also obtained from this database (ID: AY173951). To obtain 3D structures for both proteins, R5-Gp120 and X4-Gp120, a crystal structure of Gp120 obtained from Protein Data Bank (PDB ID: 2B4C) was used as a template. Homology models were built using MODELER package,33 using pairwise alignment services provided by The European Bioinformatics Institute (EMBL-EBI) (http://www. ebi.ac.uk/), considering 87.2% 2B4C sequence identity to R5-Gp120 and 81.6% 2B4C sequence identity to X4-Gp120. Human T-cell surface glycoprotein CD4 was obtained from the same crystal used as a template for Gp120 (2B4C). To obtain the conformation for Gp120-CD4 complexes, each model X4-Gp120 or R5-Gp120 was overlapped on the Gp120 crystal structure contained in 2B4C. G2-S16 Dendrimer Model. G2-S16 dendrimer structure was built in full atomic detail, by splitting it into fragments: one core, two monomers, and one terminal group, as described in a previous work performed by our group.34 Briefly, each part was parametrized under CHARMM General force field philosophy,35 by using PARAMCHEM platform and the Force Field Toolkit plugin (ffTK)36 belonging to VMD software.37 I


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prevents HIV-1 gp120IIIB binding to the cell surface. Proc. Natl. Acad. Sci. U. S. A. 111, E1960−9. (4) Nankya, I. L., Tebit, D. M., Abraha, A., Kyeyune, F., Gibson, R., Jegede, O., Nickel, G., and Arts, E. J. (2015) Defining the fitness of HIV-1 isolates with dual/mixed co-receptor usage. AIDS Res. Ther. 12, 34. (5) Delwart, E. L., Mullins, J. I., Gupta, P., Learn, G. H., Jr., Holodniy, M., Katzenstein, D., Walker, B. D., and Singh, M. K. (1998) Human immunodeficiency virus type 1 populations in blood and semen. J. Virol. 72, 617−23. (6) Wenz, W., Noeldge, G., and Grosshans, U. M. (1979) [X-ray examination for the detection of complications after abdominal surgery (author’s transl)]. Prakt. Anaesth. 14, 138−47. (7) Zhu, T., Mo, H., Wang, N., Nam, D. S., Cao, Y., Koup, R. A., and Ho, D. D. (1993) Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science 261, 1179−81. (8) Galli, G., Annunziato, F., Cosmi, L., Manetti, R., Maggi, E., and Romagnani, S. (2001) Th1 and th2 responses, HIV-1 coreceptors, and HIV-1 infection. J. Biol. Regul. Homeost. Agents 15, 308−13. (9) Nardacci, R., Perfettini, J. L., Grieco, L., Thieffry, D., Kroemer, G., and Piacentini, M. (2015) Syncytial apoptosis signaling network induced by the HIV-1 envelope glycoprotein complex: an overview. Cell Death Dis. 6, e1846. (10) Murooka, T. T., Sharaf, R. R., and Mempel, T. R. (2015) Large Syncytia in Lymph Nodes Induced by CCR5-Tropic HIV-1. AIDS Res. Hum. Retroviruses 31, 471−472. (11) Symeonides, M., Murooka, T. T., Bellfy, L. N., Roy, N. H., Mempel, T. R., and Thali, M. (2015) HIV-1-Induced Small T Cell Syncytia Can Transfer Virus Particles to Target Cells through Transient Contacts. Viruses 7, 6590−603. (12) Sepulveda-Crespo, D., Lorente, R., Leal, M., Gomez, R., De la Mata, F. J., Jimenez, J. L., and Munoz-Fernandez, M. A. (2014) Synergistic activity profile of carbosilane dendrimer G2-STE16 in combination with other dendrimers and antiretrovirals as topical antiHIV-1 microbicide. Nanomedicine 10, 609−18. (13) Cena-Diez, R., Vacas-Cordoba, E., Garcia-Broncano, P., de la Mata, F. J., Gomez, R., Maly, M., and Munoz-Fernandez, M. A. (2016) Prevention of vaginal and rectal herpes simplex virus type 2 transmission in mice: mechanism of antiviral action. Int. J. Nanomed. 11, 2147−62. (14) Sepulveda-Crespo, D., Cena-Diez, R., Jimenez, J. L., and Angeles Munoz-Fernandez, M. (2017) Mechanistic Studies of Viral Entry: An Overview of Dendrimer-Based Microbicides As Entry Inhibitors Against Both HIV and HSV-2 Overlapped Infections. Med. Res. Rev. 37, 149−179. (15) Sepulveda-Crespo, D., Jimenez, J. L., Gomez, R., De La Mata, F. J., Majano, P. L., Munoz-Fernandez, M. A., and Gastaminza, P. (2017) Polyanionic carbosilane dendrimers prevent hepatitis C virus infection in cell culture. Nanomedicine 13, 49−58. (16) Sepulveda-Crespo, D., Serramia, M. J., Tager, A. M., Vrbanac, V., Gomez, R., De La Mata, F. J., Jimenez, J. L., and Munoz-Fernandez, M. A. (2015) Prevention vaginally of HIV-1 transmission in humanized BLT mice and mode of antiviral action of polyanionic carbosilane dendrimer G2-S16. Nanomedicine 11, 1299−308. (17) Cena-Diez, R., Garcia-Broncano, P., Javier de la Mata, F., Gomez, R., Resino, S., and Munoz-Fernandez, M. (2017) G2-S16 dendrimer as a candidate for a microbicide to prevent HIV-1 infection in women. Nanoscale 9, 9732−9742. (18) Garcia-Broncano, P., Cena-Diez, R., de la Mata, F. J., Gomez, R., Resino, S., and Munoz-Fernandez, M. A. (2017) Efficacy of carbosilane dendrimers with an antiretroviral combination against HIV-1 in the presence of semen-derived enhancer of viral infection. Eur. J. Pharmacol. 811, 155−163. (19) Galan, I., Jimenez, J. L., Gonzalez-Rivera, M., De Jose, M. I., Navarro, M. L., Ramos, J. T., Mellado, M. J., Gurbindo, M. D., Bellon, J. M., Resino, S., et al. (2004) Virological phenotype switches under salvage therapy with lopinavir-ritonavir in heavily pretreated HIV-1 vertically infected children. AIDS 18, 247−55.

systems considering G2-S16 dendrimer. Then, the gp120-CD4 binding free energy, ΔGbinding, was calculated using the Molecular Mechanics/Generalized Born surface area method (MM-GBSA)45 as previously reported,34,46,47 considering the follow equation: ΔG binding = GTOTAL(gp120 − CD4complex) − (GTOTAL(gp120) + GTOTAL(CD4))GTOTAL = HMM + Gsolv − T ΔSconf

where HMM contribution was calculated by summing the in vacuo gas-phase nonbond energies (HMM = EvDW + Eele) and the solvation free energies ((Gsol) = GGB + GNP) for each component, e.g., gp120-CD4 complex, gp120 protein, and CD4 protein. Polar solvation free energy GGB was calculated using Generalized Born approach and nonpolar solvation GNP was calculated as γ (SASA) + β, in which γ = 0.00542 kcal/Å2 and β = 0.92 kcal/mol.48 The conformational entropy was not included, because of the large low prediction accuracy and computational cost. ΔΔGbinding was obtained as a difference between ΔGbinding in bound-dendrimer and unbound-dendrimer state.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] or mmunoz.hgugm@ salud.madrid.org. Telephone: +34 915 868 565. ORCID

Rafael Gomez: 0000-0001-6448-2414 M. Angeles Muñoz-Fernández: 0000-0002-0813-4500 Author Contributions

⬢ Guerrero-Beltran and Rodriguez-Izquierdo contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been (partially) funded by the RD12/0017/0037, RD16/0025/0019, projects as part of the Acción Estratégica ́ en Salud, Plan Nacional de Investigación Cientifica, Desarrollo e Innovación Tecnológica (2008−2011; 2013−2016) and cofinanced by the Instituto de Salud Carlos III (Subdirección General de Evaluación) and Fondo Europeo de Desarrollo Regional (FEDER), RETIC PT13/0010/0028, Fondo de Investigacion Sanitaria (FIS) (grant number PI16/01863), CYTED 214RT0482, EPIICAL Project and MINECO CTQ-2014-54004P. CIBER-BBN is an initiative funded by the VI National R&D&I Plan 2008−2011, Iniciativa Ingenio 2010, the Consolider Program, and CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

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REFERENCES

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