Based Microbicides As Entry Inhibitors Against Both HIV

Mechanistic Studies of Viral Entry: An Overview of Dendrimer-Based Microbicides As Entry Inhibitors Against Both HIV and HSV-2 Overlapped Infections Daniel Sepúlveda-Crespo,1,2,3 Rafael Ceña-D´ıez,1,2,3 José Luis Jiménez,2,3,4 ´ Muñoz-Fernández1,2,3 and Ma Angeles 1 Laboratorio

InmunoBiolog´ıa Molecular, Hospital General Universitario Gregorio Maran˜ on, ´ Madrid, Spain de Investigacion ´ Sanitaria Gregorio Maran˜ on ´ (IiSGM), Spanish HIV-HGM BioBank, Madrid, Spain 3 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain 4 Plataforma de Laboratorio, Hospital General Universitario Gregorio Maranon, Madrid, Spain ˜´

2 Instituto

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/med.21405

䉲 Abstract: This review provides an overview of the development of different dendrimers, mainly polyanionic, against human immunodeficiency virus (HIV) and genital herpes (HSV-2) as topical microbicides targeting the viral entry process. Vaginal topical microbicides to prevent sexually transmitted infections such as HIV and HSV-2 are urgently needed. To inhibit HIV/HSV-2 entry processes, new preventive targets have been established to maximize the current therapies against wild-type and drug-resistant viruses. The entry of HIV/HSV-2 into target cells is a multistep process that triggers a cascade of molecular interactions between viral envelope proteins and cell surface receptors. Polyanionic dendrimers are highly branched nanocompounds with potent activity against HIV/HSV-2. Inhibitors of each entry step have been identified with regard to generations and surface groups, and possible roles for these agents in antiHIV/HSV-2 therapies have also been discussed. Four potential binding sites for impeding HIV infection (HSPG, DC-SIGN, GSL, and CD4/gp120 inhibitors) and HSV-2 infection (HS, gB, gD, and gH/gL inhibitors) exist according to their mechanisms of action and structures. This review clarifies that inhibition of HIV/HSV-2 entry continues to be a promising target for drug development because nanotechnology can transform the field of HIV/HSV-2 prevention by improving the efficacy of the currently available C 2016 Wiley Periodicals, Inc. Med. Res. Rev., 00, No. 0, 1–31, 2016 antiviral treatments. Key words: HIV/HSV-2 entry inhibitors; mechanism of antiviral action; microbicide; polyanionic dendrimer; sexually transmitted infections ´ Correspondence to: Ma Angeles Munoz-Fern andez, Laboratorio InmunoBiolog´ıa Molecular, Hospital General ˜ ´ Universitario Gregorio Maran˜ on, ´ C/Dr. Esquerdo 46, 28007 Madrid, Spain. E-mail: [email protected] Medicinal Research Reviews, 00, No. 0, 1–31, 2016 C 2016 Wiley Periodicals, Inc.

2

´ r SEPULVEDA-CRESPO ET AL.

1. INTRODUCTION Sexual contact is the primary mode of human immunodeficiency virus (HIV) transmission. Herpes simplex virus type 2 (HSV-2) is among the most common sexually transmitted infections (STIs) and is the leading cause of genital ulcers worldwide. HSV-2 is widespread even among people with low intercourse frequency, and, importantly, most people are not aware of being infected. HSV-2 infection has received more attention over the years because the disease burden is high and because of a better understanding of the synergy between HSV-2 and HIV. In fact, several studies have shown that HSV-2 genital ulceration is associated with a two- to fourfold increased risk of acquiring HIV infection.1, 2 The presence of HSV-2 enhances HIV susceptibility by disrupting the epithelial surface, recruiting HIV target cells to the genital tract and/or by producing a proinflammatory local milieu.3 Potential strategies (i.e., dual microbicides or suppressive therapies) that could reduce or eradicate genital HIV/HSV-2 co-infection should be explored. Despite considerable efforts to develop a vaccine against HIV and HSV-2, effective vaccines to prevent the spread of HIV/HSV-2 are not yet available.4–6 Therefore, effective prevention strategies, including dual microbicides for treating HIV/HSV-2 co-infection, are needed to attain the goals of the WHO and UNAIDS: zero new infections, zero discrimination, and zero deaths. The viral entry of HIV and HSV-2 is a multistage process that involves sequential interactions between glycoproteins of the virions and various surface receptors of the host cells. This review evaluates the structures of the main HIV and HSV-2 surface proteins, the mechanisms of viral entry into cells, and the development of different dendrimers, mainly polyanionic, to inhibit specific steps in HIV/HSV-2 entry.

2. THE HIV ENTRY PROCESS CD4+ T-lymphocytes, monocyte/macrophage lineage cells, and dendritic cells (DCs) are the main target cells of HIV.7 Before establishing an infection per se in CD4+ T-cells, the virus can interact with alternative receptors that facilitate HIV infection.8–10 Contact between HIV and target cells is accomplished by specific or nonspecific interactions between HIV proteins and their ligands on the surfaces of host cells. A specific attachment can result from Env/α4β7 integrin interactions, intercellular adhesion molecule 1 (ICAM-1)/lymphocyte function-associated antigen 1 (LFA-1) interactions, or carbohydratebinding proteins, such as DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN).11, 12 Nonspecific attachment results from electrostatic interactions between positively charged domains on HIV gp120 and negatively charged heparan-sulfate proteoglycans (HSPGs) or glycosphingolipids (GSLs) on target cells.13, 14 Although these interactions (especially with HSPGs, which are present on the surfaces of practically all cell types), do not allow for productive infection, they play major roles in diffusion, attachment, and cellular entry.15 Once the virus is close to the cell membrane, binding of the HIV gp120 to the CD4 receptor of the cell surface and various conformational changes in gp120 occur. First, the D1 domain of CD4 joins the CD4-binding site of gp120, which is a highly conserved carbohydrate-free region.16 Then, the V1/V2 domain of gp120 modifies its position and its flexibility, and the V3 loop extends away from the virion spike and is positioned toward the cell membrane to interact with CCR5 or CXCR4 co-receptors.17–19 Therefore, the V3 loop is the main domain involved in this interaction, and V3 amino sequences determine the co-receptors used by HIV to enter host cells.20, 21 Medicinal Research Reviews DOI 10.1002/med

MECHANISTIC STUDIES OF VIRAL ENTRY

r 3

Binding of gp120 to co-receptors involves two sequential interactions. The N-terminus of CCR5 or CXCR4 binds to the V3 loop, altering the conformation of the V3 loop and facilitating the second interaction with extracellular loops of co-receptors, which are critical for HIV entry.19, 22 The proximity between cellular and viral membranes allows for gp41-mediated membrane fusion and the entry of the viral capsid into host cells.23 Although the events that lead to HIV entry occur at the cell surface, endocytosis can emerge as an alternative entry route for HIV.24–26 This aspect is important for the consideration of novel entry inhibitors because effective concentrations of these compounds can be more difficult to attain in endosomal compartments. 3. THE HSV-2 ENTRY PROCESS HSV-2 entry into host cells is mediated and modulated by the action of seven glycoproteins through their interactions with their cognate receptors: gB, gC, gD, gH, gK, gL, and gM. However, only five glycoproteins (gB, gC, gD and the heterodimer gH/gL) play important roles in HSV-2 fusion between the viral envelope and the membrane of host cells.27–30 The first step in HSV-2 entry is the binding of the virus through gB and gC to HSPGs on the cell surface to tether the virus at the cell surface. gB can perform this function in the absence of gC, although it reduces HSV-2 efficiency.31 The next step is the specific interaction between gD and multiple receptors, such as the herpes virus entry mediator (HVEM), nectin-1, nectin-2, and, to a lesser extent, 3-O-sulfated HSPG.32 The interaction of gD with one of its receptors causes conformational changes and transmits an activation signal to gH/gL (acting as a regulator of the fusion process) in a form that binds to gB to start the viral-membrane fusion.33, 34 Finally, the fusion between the viral envelope and the membrane of host cells releases the viral nucleocapsid into the cell cytoplasm.28 4. VAGINAL TOPICAL MICROBICIDES Efforts to prevent the heterosexual transmission of HIV/HSV-2 have been based on three approaches: behavioral changes (safer sex), the development of vaccines, and the development of dual microbicides. Abstinence and the use of condoms to reduce the risk of cross-sex transmission have not been successful for reasons of financial insecurity, fear of retaliation, desire for pregnancy, or avoidance of sex. Abstinence and the use of condoms reduce the rate of transmission by only approximately 50%, and they do not avoid the increased risk of HIV acquisition or transmission in HSV-2-seropositive people. Advances in vaccines have been disappointing, and topical dual microbicides provide excellent potential for a female-controlled, preventive option that does not require negotiation, consent, or even their partner knowing. Thus, a dual microbicide is likely to become available more rapidly than a vaccine because it can be used by both men and women. Microbicides are compounds formulated as gels, creams, films, or suppositories that are applied inside the vagina to protect against HIV/HSV-2 and other STIs. Cell-free HIV/HSV-2 can enter the vaginal epithelium by diffusing across a concentration gradient (transcytosis), and is trapped on the surfaces of epithelial cells until HIV can be taken up by intra-epithelial Langerhans cells. The successful transfer of HIV results in HIV uptake by DCs in the subepithelium and subsequent dissemination to draining lymph nodes. HIVinfected cells can also cross the epithelial barrier by physical abrasion or by transmigration. By contrast, HSV-2 spreads and enters sensory neurons, where it establishes a latent infection and forms a reservoir for a recurrent infection, disease, and transmission to other people (Fig. 1).35, 36 Medicinal Research Reviews DOI 10.1002/med

4


Figure 1. Potential modes of action for dual microbicides to reduce the risk of HIV/HSV-2 transmission. The microbicide must act as a lubricant coat, providing a physical barrier against HIV-1 and other sexually transmitted infections (STIs), such as HSV-2. Once HIV/HSV-2 has crossed the epithelial barrier, the microbicide must prevent HSV-2 from entering the sensory neurons and establishing latency, HIV uptake by dendritic cells (DCs), HIV attachment, and fusion and/or block HIV replication before integration. The dual microbicide must prevent HIV from entering by physical abrasion, direct infection, transcytosis, or uptake by Langerhans cells.

Some characteristics of an ideal vaginal microbicide include the following: display activity against most HIV/HSV-2 strains and other STIs; act as a direct virucidal agent; retain activity for several hours in the presence of vaginal fluids and semen and over a broad pH range; not disrupt the normal vaginal flora and the structural integrity of the vaginal mucosal epithelium (Fig. 1); be odorless, colorless, tasteless, stable at higher temperatures, and easy to use; have a long shelf-life; and be inexpensive, readily accessible, and compatible for use with latex, among other qualities.37–40

5. DENDRIMERS The application of nanomedicine has resulted in the development of new structures, complexes, and nanotechnological composites, such as dendrimers. Dendrimers are highly branched, starshaped, and nano-sized molecules defined by three components: a nucleus, scaffold layers or generations, and functional groups at the outer surface (Fig. 2). Dendrimers have diameters ranging from 1 to 40 nm, and they adopt a more globular shape with increasing generations.41–43 The diameter of dendrimers increases linearly on the order of 1 nm/generation, and the number of surface groups increases exponentially with each generation. The synthesis, physicochemical characterization, and other specific properties of dendrimers have been previously reported widely.44–47 A. Properties of Dendrimers Dendrimers are macromolecules with roughly spherical or globular shapes. The globular shape and size of dendrimers allow them to mimic biological species and to establish some analogies Medicinal Research Reviews DOI 10.1002/med


r 5

Figure 2. Schematic representation of the general structure of a dendrimer. Dendrimers are divided into three architectural components: an initiator core, interior layers with repeating units or generations, and an exterior with functional end-groups.

with globular proteins, which would explain their great impact and the substantial research interest in their biomedical applications.48–50 The size and molecular weight of dendrimers are obtained and controlled in a careful stepwise manner, with branching units increasing exponentially from a central core to the surface of the dendrimer. The size control is important in biomedical applications because diverse molecular sizes exhibit different pharmacokinetics. Dendrimers are well-organized molecules with exactly the same molecular weight and structure and with very low polydispersity due to careful synthetic and purification procedures. In biomedicine, the monodispersity must prevail over the high cost of multistage synthesis. In solution, dendrimers form a tightly globular structure that has an enormous impact on their rheological properties. The intrinsic viscosity of dendrimers does not increase linearly with their molecular weight, but it achieves a maximum value and then starts to decline because lower generations of dendrimers adopt a more planar shape with higher molecular surface to volume ratios, whereas higher generations adopt a more compact spherical shape. Most of the physicochemical properties of dendrimers depend on functional end-groups that are responsible, for instance, for high solubility and reactivity. The presence of several end-groups in dendrimers enables versatile functionalization, known as multivalency. There has been growing interest in designing potent multivalent compounds that influence biological interactions to inhibit undesired biological interactions, promote desired cellular responses, and control recognition at the surface. Viruses use multivalent interactions to bind to receptors with increased avidity and specificity. Therefore, multivalent dendrimers can be used as inhibitors of the attachment of viruses to the receptors of target cells. The major advantage of dendrimer multivalency is that multivalent interactions are collectively much stronger than corresponding monovalent interactions.51–53 Medicinal Research Reviews DOI 10.1002/med

6


Figure 3. Potential applications of dendrimers as topical microbicides according to different targeting sites against HIV and HSV-2. Dendrimers as HIV entry inhibitors blocking gp120/CD4, gp120/DC-SIGN, gp120/glycosphingolipid (GSL), or gp120/heparan-sulfate proteoglycans (HSPGs) interactions. Dendrimers as HSV-2 entry inhibitors blocking attachment and adsorption of HSPGs or the gH protein. PLL, poly-L-lysine dendrimer; PPI, polypropylenimine dendrimer; PAMAM, polyamidoamine dendrimer; PPH, phosphorus-containing dendrimers.

B. Types of Dendrimers Tested As Vaginal Topical Microbicides: An Overview of Structure–Activity Relationships (SARs) To date, several dendrimers with diverse architectures and end-groups have been designed for use as topical microbicides against HIV and HSV-2 (Fig. 3). Some of the most important dendrimers (anionic, neutral, or modified dendrimers) include the following: polyamidoamine (PAMAM) dendrimers, poly-ι-lysine (PLL) dendrimers, polypropylene-imine (PPI) dendrimers, phosphorus-based (PPH) dendrimers, carbosilane dendrimers, metallodendrimers, dendritic structures based on Boltorn hyperbranched polymers, peptide dendrimers, glycodendrimers, and amphiphilic dendrimers. The type of core, generations, and different end-groups of dendrimers impart various biological and pharmacological properties with broad-spectrum antiviral activity. The SARs of different dendrimers with regard to HIV and HSV-2 inhibitory activity are functions of mainly five factors: various targets involved in the viral entry; the kinetics of viral entry; the number of generations; surface groups; and, to a lesser extent, the type of core of the Medicinal Research Reviews DOI 10.1002/med


r 7

dendrimers. Viruses rely on interactions with host receptors for binding and entry into target cells. Productive HIV entry is mainly dependent on gp120 binding to CD4 and CCR5/CXCR4 co-receptors (see Section 2), whereas HSV-2 entry relies on the interactions of gB and gC with HSPG and of gD with HVEM, nectin-1, nectin-2, or 3-O-sulfated HSPG, as well as the fusion regulated by gH/gL (see Section 3). Therefore, differences in the SARs of the same dendrimer for HIV and HSV-2 are likely due to different targets involved in viral entry. The SARs of dendrimers with different architectures do not follow a fixed rule and are not always consistent (i.e., PAMAM vs. PLL). However, the SARs of dendrimers within the same group (the same structural design, e.g.., PAMAM) with regard to the number of generations, surface groups, or cores can be engineered for optimal inhibition of HIV, HSV-2, and even other STIs. Generations and end-groups of dendrimers are interconnected because the number of surface functional groups increases exponentially with each ensuing cycle or generation, and the maximum size or generation of a dendrimer is governed by steric hindrance of end-groups. Lower generations of dendrimers are more flexible and tend to be more open and amorphous structures than dendrimers with higher generations, which can adopt spherical conformations. The different results in the SARs of flexible and rigid dendrimers are due to the flexibility of dendrimer arms, the core, the dendrimers being more constrained spatially and different end-groups being able to interact with each other.

6. HIV ENTRY-INHIBITING DENDRIMERS AS MICROBICIDES HIV entry and subsequent infection rely on a complex sequence of steps involving interactions between the proteins of HIV and host surface proteins. There are several targets that impede the HIV entry process. Polyanions used systemically and as vaginal microbicides showed a lack of efficacy and even enhancement of HIV infection,54 clearing the way for novel potent HIV entry inhibitors with unique characteristics to prevent sexual transmitted HIV. Dendrimers as HIV entry inhibitors are a heterogeneous group of compounds with multiple mechanisms of action. In this review, we mainly focus on polyanionic dendrimers that have been tested and their modes of action are known according to potential binding sites (Table I). A. Dendrimers Blocking Heparan-Sulfate Proteoglycans/gp120 Interactions HSPGs are glycoproteins that contain one or more covalently attached HS glycosaminoglycan chains (Fig. 4A).55 HIV can use HSPGs to transit from the extracellular environment to the cell interior. The membrane-penetrating peptide HIV-tat is released from HIV-infected cells and enters surrounding cells using HSPGs.15, 56, 57 Peptide dendrimers contain a peptidyl branching core covalently attached to basic amino acids (aas) that can bind to the negatively charged sulfate and carboxyl groups of HS.58 Therefore, dendrimeric cell-penetrating peptides rich in arginine or lysine residues that facilitate interactions with HSPGs should be prepared to prevent HIV infection.59 Two peptide dendrimers on a lysine core with the peptide sequence ASLRVRIKK (SB105A10) or NKKIRVRL (SB104) were synthesized as molecular antagonists of HSPG. Both peptide dendrimers possessed the same net positive charge with four basic aas. SB105-A10 exhibited antiviral activity against several HIV-1 isolates from HIV+ individuals in peripheral blood mononuclear cells (PBMCs), and it prevented HIV attachment/entry by multiple mechanisms targeting HSPGs and HIV virions, specifically by binding to gp41 and gp120. However, SB104 did not show anti-HIV activity, and this effect was attributed to different sequences in the external peptide chains.60 This study showed that basic aas of SB105-A10 could bind to the negatively charged sulfated or carboxyl groups of the HS chain of HSPGs, especially in HS in Medicinal Research Reviews DOI 10.1002/med

Boltorn Boltorn

Glycodendrimer PAMAM PPI

Boltorn PPH

PPH

PLL

PLL

PPI PPI PAMAM PAMAM

PAMAM

PLL

PAMAM

Bol13.4 LewisX -PAMAM PSGal64mer

BH3OPSGal PPH-3d-G1

PPH-5c-Gc’1

SCSLD3

PLDG3-PSCel

MVC-GBT MVC-3SL Sulfo-6 SPL2923

SPL6195

SPL7013

SPL7304

Classification dendrimer

BH30sucMan Dendron12

Code name

Medicinal Research Reviews DOI 10.1002/med G4

G4

G4

G5 G5 G2 G4

G3

G3

G1

G3 G1

G1 G5 G5

G3 G3

Generation

Benzhydrylamine amide


Ethylendiamine

Benzhydrylamine amide 1,4-Diaminobutane 1,4-Diaminobutane Ethylendiamine Ammonia

Stearylamide

Cyclotriphosphazene

Bis-MPA Cyclotriphosphazene

Bis-MPA 3-Azidopropanoic acid Pentaerythritol Ethylendiamine 1,4-diaminobutane

Core

32

32

32

46 28 16 11 24

24

32

12

32 12

6 14–16 44 2

32 4

Number of end-groups

1-(Carboxymethoxy) naphthalene-3,6disulfonate 1-(Carboxymethoxy) naphthalene-3,6disulfonate

Pseudo-dimmanoside Glycan Galactose sulfate per galactose residue β-Galceramide Galactosylceramide, Nhexadecylaminolactitol Phosponic acid moiety and lateral alkyl chain Cellobiose degree of sulfation: 2.3 Cellobiose degree of sulfation: 1.9 Globotriose 3 Sialyllactose Sialic acid sulfate 1-(Carboxymethoxy) naphthalene-3,6disulfonate Benzene dicarboxylate

Mannose Pseudo-trimannoside

Functional end-groups

Targeting sites

Continued

GSL GSL GSL gp120 binding/fusion/ RT/IG gp120 binding/fusion gp120 binding/fusion/ RT/IG gp120 binding/fusion/ RT/IG

GSL

GSL

GSL

GSL GSL

DC-SIGN DC-SIGN GSL

DC-SIGN DC-SIGN

Table I. Characteristics of Dendrimers With Different Functional Groups As Vaginal Topical Microbicides and the Main Sites Targeting HIV

8 ´ r SEPULVEDA-CRESPO ET AL.

G2 G2 G1

Viologen

PPI

PPI

Peptide dendrimer

Viol7

Metallo-dendrimer G2S Metallo-dendrimer G2C Trp(5a-5f) Carboxylic acid with an aminotriester as a branching unit

Ethylenediamine

Ethylenediamine

Benzyl

Benzyl

Silicon Silicon Silicon Silicon Silicon Polyphenolic Gallic acid-triethylene glicol Benzhydrylamine amide



Core

9-18

16

16

6

6

4

16 16 16 16 16 24 9

32

32

Number of end-groups

Tryptophan

Carboxylate

Sulfonate

Thymine

Ethyl

Sequence peptide chain (ASLRVRIKK)

1-(Carboxymethoxy) naphthalene-3,6disulfonate 1-(Carboxymethoxy) naphthalene-3,6disulfonate Sulfonate Sulfonate Carboxylate Naphthylsulphonate Sulphate Sulfonate Benzoate


gp120 binding/fusion

gp120/CD4

HSPG /gp120 binding/fusion CXCR4 antagonist CXCR4 antagonist gp120/CD4

gp120/CD4 gp120/CD4 gp120/CD4 gp120/CD4 gp120/CD4 gp120/CD4 Assembly

gp120 binding/fusion/ RT/IG gp120 binding/fusion

Targeting sites

Bis-MPA, 2,2-bis(hydroxymethyl)propionic acid; RT, reverse transcriptase; IG, integrase; GSL, glycoshpingolipid; HSPG, heparan-sulfate proteoglycan.

G1

G1

Viologen

G1

G1 G2 G2 G1 G2 G2 G1

Viol36

Carbosilane Carbosilane Carbosilane Carbosilane Carbosilane Carbosilane GATG

G2-S16 G2-STE16 G2-CTE16 G1-NS16 G2-Sh16 G2-S24P [G1]-CO2Na

G2

Peptide dendrimer

PPL

SPL7115

G4

Generation

SB105-A10

PPI


SPL7320

Code name

Table I. Continued


r 9

Medicinal Research Reviews DOI 10.1002/med

10


Figure 4. Heparan-sulfate proteoglycan structural characteristics and the mechanism of action of peptide dendrimers in preventing HIV infection. (A) HSPGs are composed of a core and one or more heparan-sulfate glycosaminoglycan chains assembled on the core. (B–E) Antagonist peptide dendrimers can act in different steps considering that HS plays many roles in the capture of HIV: (B) to kidnap HIV from nonpermissive cells (epithelial cells) and mediate infection by presenting the virus to permissive cells (DCs and CD4+ T-cells); (C) to facilitate the transcytosis of HIV through the epithelium; (D) to capture viral particles and facilitate subsequent interactions with specific entry receptors; and (E) to facilitate interactions among amyloid fibrils, HIV, and target cells.15, 61, 62

the cell membranes of activated PBMCs. Using surface plasmon resonance (SPR) assay, the authors demonstrated that SB105-A10 also bound to gp41 with high affinity and, to a lesser extent, to gp120. It is reasonable to believe that SB105-A10 can alter the correct steric interactions between both viral glycoproteins and cellular receptors due mainly to the polyvalency of the peptide dendrimer. Moreover, the SPR assay showed that the SB105-A10/gp41 interaction was localized to the ectodomain of gp41 involved in the formation of the bundle and in the fusion between the virus and the cell surface (region of aas 546–682). In summary, peptide dendrimers must be able to avoid HS to capture HIV and facilitate the subsequent interaction with CD4 and CCR5/CXCR4, to trap HIV from epithelial cells mediating infection by presenting HIV to DC and CD4+ T-cells, to facilitate transcytosis of HIV through the epithelium, and to facilitate interactions among amyloid fibrils, HIV, and host cells (Fig. 4B).15, 61, 62 B. Dendrimers Blocking gp120/DC-SIGN Interactions DC-SIGN is a tetrameric calcium-dependent (C-type) lectin that interacts mainly with highmannose glycans, which are the branched oligosaccharides present in multiple copies among Medicinal Research Reviews DOI 10.1002/med


r 11

Figure 5. Structure, function of DC-SIGN, and mechanism of action of glycodendrimers in preventing dendritic cell-mediated HIV transmission. (A) Domain organization of DC-SIGN.63 DC-SIGN is composed of four domains: (i) a C-terminal carbohydrate recognition domain; (ii) a neck region composed of 7 and a half repeats of 23 aa residues; (iii) a transmembrane domain; and (iv) a N-terminal cytoplasmic tail. (B) Glycodendritic structures can stop the DC-mediated HIV trans-infection by avoiding DC-SIGN: (i) the infectious synapse where DC transfer captures HIV to CD4+ T-cells, or (ii) the release of HIV-associated exosomes that are transmitted to CD4+ T-cells through membrane binding and fusion.69 (C) Representation of DC-SIGN/glycodendritic structure interaction affinity: (i) weak interactions by poorly expressed, low density DC-SIGN, and (ii) strong binding due to high density DC-SIGN generated through overexpression and oligomerization.82

several pathogens (Fig. 5A).63, 64 DC-SIGN is a molecule expressed on the surface of immature DCs and involved in the early stages of HIV infection, interacting with high-mannose glycans at gp120 and facilitating its dissemination through the trans- and cis-mechanisms.65–69 DC-SIGN can also recognize branched fucosylated structures bearing terminal fucose residues (i.e., Lewis antigens). Therefore, the inhibition of the gp120/DC-SIGN interaction is considered a strategy to design novel dendrimers with antiviral activity. In this sense, highly mannosylated or fucosylated multivalent glycodendritic structures on adequate scaffolds mimicking the N-linked high-mannose or fucosylated carbohydrates arrangement on gp120 have played key roles.70–74 Polyguanidylated glycodendrons with 3, 9, and 27 copies of linear tetramannosyl ligand (Man4 ), corresponding to the arm of mannose or a branched nanomannosyl (Man9 ) that represents high-mannose glycan, were demonstrated to inhibit binding between DC-SIGN and gp120 due to oligomannose dendrons interacting with DC-SIGN on the cell surface.75 Boltorn hyperbranched dendritic polymers based on 2,2-bis(methylol)propionic acid (bisMPA) as the building block were also assessed for inhibition of HIV-1 entry with DC-SIGN as the target. Glycodendritic structures based on Boltorn hyperbranched polyesters polymers are Medicinal Research Reviews DOI 10.1002/med

12


easy to synthesize from a bis-MPA monomer and ethoxylated pentaerythritol as a core, and they are perfectly soluble under physiological conditions.76 The ability to bind to the second and third generations of Boltorn dendritic polymers functionalized with 16 and 32 mannoses to DC-SIGN occurs through the carbohydrate recognition domain (CRD) of lectin by the coordination of mannoses to a calcium-binding site exposed to the surface of the protein. The third generation conjugated with 32 mannoses binds to DC-SIGN with greater affinity than the second generation conjugated with 16 mannoses.77 Improvement of these antiviral activities of Boltorn-type dendrimers with a simple carbohydrate (a mannose monosaccharide) was performed using more complex and specific carbohydrate ligands. The synthesis of two glycomimetic compounds, based on the tetravalent glycodendron and a third generation Boltorntype dendrimer with four copies of pseudo-disaccharide 1,2-mannobioside (containing a mannose connected to a conformationally locked cyclohexanediol to mimic 1,2-mannobioside), reduced HIV trans-infection of CD4+ T-cells by more than 90% at 50 μM and 95% inhibition at 100 μM.78, 79 To optimize pseudo-disaccharide structures and to improve the selectivity of binding to DC-SIGN, new glycodendrimers with polyalkenes as cores were synthesized. A hexavalent presentation with linear pseudo-dimannoside groups at the periphery displayed high potency blocking of the gp120/DC-SIGN interaction and inhibited the HIV trans-infection of CD4+ T-cells by 100% at 10 μM, as well as improving accessibility and chemical stability relative to the aforementioned dendrimers.80 On the basis of the glycan specificity of C-type lectin receptors, Lewis-type antigens are interesting candidates for the design of DC-SIGN-specific antagonists. PAMAM dendrimers consist of an ethylenediamine core, a backbone with repeated branching units of amidoamine and a primary amine terminal surface. Lewis-type PAMAM dendrimers are built using PAMAM dendrimers by the conjugation of different glycans through their reducing end to end-groups of dendrimers. Several generations of fucosylated Lewis-type PAMAM glycodendrimers, differing in the number of functional groups from 16 (third generation) to 64 (fifth generation) and with average glycan units in the range of 14–16 per glycodendrimer, were shown to be effective in blocking gp120/DC-SIGN binding. Generation 5 LewisX (lacto-N-fucopentaose III)-type PAMAM glycodendrimer was the most effective in blocking gp120/DC-SIGN binding with high selectivity and avidity and in reducing the transmission of HIV to CD4+ T-cells by nearly 100% at submicromolar concentrations. The main conclusions of this study were that the multivalent character and geometry of glycodendrimers are important factors in determining their functionality.81 These glycodendritic structures block HIV captured by DC via DC-SIGN efficiently and avoid the trans-infection of CD4+ T-cells during adhesion between both cells. The transinfection can be stopped by glycodendritic structures with DC-SIGN avoiding (i) infectious synapse, or (ii) exocytic pathways that involve the HIV-associated exosomes that are transmitted to CD4+ T-cells through membrane binding and fusion (Fig. 5B).69 The high density of DC-SIGN expression on DCs results in strong binding of glycodendrimers and subsequent colonization, avoiding the capture of HIV particles by DCs. For this reason, glycodendrimer binding to DC-SIGN is multivalent. Carbohydrate epitopes on the surface of the host cells that are used by bacteria and viruses for colonization and infection constitute the starting point of the search for glycomimetic entry inhibitors. Therefore, DC-SIGN binding sites are clustered due to overexpression and oligomerization processes,82 leading to strong bonds by glycodendritic structures (Fig. 5C). These mannosyl/fucosyl functionalized hyperbranched dendritic structures open new outlooks into the design of antivirals using the DC-SIGN receptor as the target. However, glycodendritic structures are recognized and degraded by mannosyl glycosylases.72 Thus, modifications in carbohydrate units and in the nature of the linker, and further work in this direction, must be conducted to avoid hydrolytic enzymes that recognize carbohydrates. Medicinal Research Reviews DOI 10.1002/med


r 13

Figure 6. Structure, function of glycosphingolipids (GSLs), and mechanism of action of glycodendrimers. (A) General structure of GSLs involved in HIV infection. GSLs are composed of two parts: (i) the lipid moiety common to each GSL (ceramide), and (ii) different sugars extending out the membrane: galactose, 3’-sialyllactose, 3 sulfogalactose, and globotriose derivatives. (B) Glycodendrimers can be bind to V3 loops, avoiding interaction of GSLs with gp120, complicating infection by association with lipid rafts for free movement of HIV through the membrane and avoiding the binding to CXCR4/CCR5 for fusion and the subsequent viral entry.

C. Dendrimers Blocking gp120/Glycosphingolipids Interactions GSLs are components of the cell membrane composed of a membrane bound lipid (ceramide) coupled to an extracellular carbohydrate. GSLs are involved in the binding and fusion of HIV and host cell membranes. GSLs are organized in functional microdomains linked to CD4 that stabilize HIV binding to CD4 through several low-affinity interactions between the V3 loop and the carbohydrate moiety of GSLs, transmitting HIV to an appropriate coreceptor by free movements.83–87 Several GSLs are recognized by gp120, such as galactosylceramide (GalCer), 3 -sulfogalactosylceramide (SGalCer), globotriaosylceramide (Gb3), and 3 sialyllactose (GM3, Fig. 6A).13 Thus, new strategies based on glycosphingolipid mimics that recognize HIV gp120, inhibit HIV fusion and consequently alter the affinity of GSL-ligand interactions must be explored. Various multivalent glycodendrimers have been synthesized to mimic the natural clustering of carbohydrate moieties due to their propensity to form spherical nanocellular decoys. Several generations of PPI glycodendrimers functionalized with sulfated and nonsulfated galactose residues have shown efficiency against HIV infection. Glycodendrimers with specific position sulfate-modified galactose were the best candidates due to preferential interaction between specific sulfates and the V3 loop.88, 89 The synthesis of water-soluble hyperbranched dendrimers based on Boltorn H30 and functionalized with natural β-galceramide also showed binding ability to gp120. Again, different behavior in HIV inhibition was attributed to the presence of sulfate groups in the molecule. However, these Boltorn glycodendrimers revealed lower affinity for gp120 than PPI dendrimers.90 The synthesis of dendrimeric GalCer analogs based on zwitterionic assemblies of phosphonic acid-terminated dendrimers and N-hexadecylamino-lactitol Medicinal Research Reviews DOI 10.1002/med

14


moieties demonstrated low therapeutic indices that prevented them from being so-called promising candidates due to relatively high CC50 values related to a lack of stability of the assemblies.91 In this regard, the same group showed that the length of an alkyl chain located close to the phosphonic acid end-groups increased the stability of the assembly.92 Compared with glycodendrimers with specific position sulfate-modified galactose, generation 3 PLL-dendritic randomly sulfated cellobiose showed high anti-HIV activity.93 The same group synthesized the same glycodendrimer with long hydrophobic alkyl chains (amphiphilic features) in the dendrimer to immobilize the sulfated cellobiose cluster.94 However, the antiHIV activity of both glycodendrimers was similar. The main conclusions of these studies were that the compact structure and cluster effect of cellobiose glycodendrimers play important roles in anti-HIV activity. Moreover, specific position sulfation is not required for the interaction between the sulfate residues of glycodendrimers and the V3 loop. Two types of PPI glycodendrimers with GM3 and Gb3 carbohydrates as end-groups were shown to inhibit HIV infection. Gp120 recognizes these structures as ligands, disrupting lipid rafts present in the membrane that are needed for the formation of a fusion pore complex.95 In a mixture of GalCer and Gb3, gp120 preferentially interacts with Gb3, suggesting that the Gb3 binding is dominant. Thus, a good strategy would be to find glycodendrimer-sulfated-Gb3 conjugates to stop the interaction of the V3 loop with CD4 and/or co-receptors.96 To search for easy-to-make and cost-effective compounds with the same or better antiviral potency, sulfated sialic acid-terminated PAMAM glycodendrimers were synthesized. Although these compounds demonstrated high anti-HIV activity, they were less potent than the aforementioned glycodendrimers.97 Therefore, several lines of evidence have supported that GSLs are needed to trigger conformational changes in gp120 for membrane fusion.98 HIV fusion occurs in GSL-enriched domains of the membrane. Different sulfated glycodendrimers can be synthesized to act on any part of these steps: (i) interaction of GM3 and Gb3 mainly with CD4; (ii) binding of gp120 to several GSLs; (iii) fusion process assembled in GSL-enriched domains; and (iv) removal of cellular cholesterol, which renders cells resistant to HIV-mediated syncytium development (Fig. 6B and C).99, 100 Recently, the potential impact of GSLs in DC-mediated HIV transinfection was found by modulating the virus infectivity or CD4+ -T cell membranes to affect viral entry.101 By contrast, the synthesis of new glycodendrimers bearing nonrandomly sulfated carbohydrate residues should be considered because they are better inhibitors than nonsulfated glycodendrimers. The poor inhibition by glycodendrimers with nonsulfated carbohydrates was due to their inability to block the interaction of positively charged V3 loops with sulfated-amino terminus of CD4 and/or CXCR4/CCR5. D. Dendrimers Blocking gp120/CD4 Interactions Several strategies based on polyanionic dendrimers for blocking gp120/CD4 interactions have been proposed (Fig. 7A). Gp210 contains regions with essential aas that promote electrostatic binding to the cell surface. These regions are the principal neutralizing domain V3 loop (aas 303–338), the C-terminal region (aas 495–516), and conserved regions involved in co-receptor binding (aas 117–123, 207, 419–444).18, 102, 103 Polyanionic dendrimers that bind to a specific region within the CD4 binding pocket of gp120 and that block the gp120/CD4 interaction (Fig. 7B) are promising candidates as microbicides. Two polyanionic PAMAM dendrimers, fully capped on the surface with 32 phenyldicarboxylic acid groups (SPL6195) or 24 naphthyldisulfonic acid groups (SPL2923), inhibited clinical HIV-1 isolates, HIV isolates resistant to reverse-transcriptase inhibitors and several HIV-2 isolates. The antiviral activity of polyanionic PAMAM dendrimers is based on their interaction with gp120, blocking virus-cell binding. Interestingly, high concentrations of SPL2923 were Medicinal Research Reviews DOI 10.1002/med


r 15

Figure 7. Model for HIV entry considering only gp120/CD4 interaction and the mechanism of action of polyanionic dendrimers. (A) HIV contains on its surface two glycoproteins that are used for its entry into target cells: gp120 and gp41. (B) Schematic model of the multistep HIV entry process. Polyanionic dendrimers block the binding and engagement of gp120 with CD4 and avoid conformational changes in gp120 that trigger its interaction with conserved co-receptor binding sites, mainly by movement of the V3 loop. Consequently, gp41 is not exposed in a fusogenic conformational state that triggers the insertion of the fusion peptide into the membrane of the target cell in a triple-stranded coiled-coil.

taken up by host cells and blocked two more processes of HIV replication: reverse transcription and integration. The main conclusions of this study were that differences in the core (ethylene diamine for SPL6195 vs. ammonia for SPL2923) and end-groups of dendrimers had effects on the ability to curb the replication of HIV.104 PAMAM dendrimers weaken the gp120/CD4 complex and alter the dissociation pathway of the complex, hindering HIV entry into target cells. Moreover, PAMAM dendrimers modulate the interactions of hydrophobic and hydrophilic residues in gp120 with CD4, alter the hydration of the hydrophobic interfacial cavity, and disrupt hydrogen bonds between gp120 and CD4.105 Considering these promising results and the structure of SPL2923, three dendrimers (PAMAM with ethylenediamine core, PLL and PPI) of a fourth generation with naphthyldisulfonic acid groups on the surface were synthesized to enhance anti-HIV activity.106 Although the biological activity of the three modified dendrimers was practically the same, the PPI-based dendrimer (SPL7320) and PAMAM dendrimer (SPL7304) were not considered optimal candidates for clinical trials. The reasons were the level of residual cobalt in SPL7320, the risk of controlling this variable on a large scale, and the risk of reverse Michael reaction in acidic formulation in the synthesis of SPL7304. A PLL-based dendrimer (SPL7013) showed stability and potential for large-scale synthesis. SPL7013 was selected for comparison with a dendrimer of the same family, a second generation, comprising a benzhydryl amide core PLL dendrimer (SPL7115).107 SPL7013 and SPL7115 also inhibited HIV reverse transcription, although SPL7115 was less effective. However, SPL7013 and SPL7115 did not inhibit reverse transcription in HIV-infected cells, most probably because they failed to enter cells or could not access reverse transcriptase within the reverse transcription complex. SPL7013 and SPL7115 blocked HIV envelope-mediated cell-to-cell fusion, although SPL7013 was more potent in inhibiting envelope-mediated fusion. Molecular modeling indicated the potential for electrostatic interactions of both dendrimers with cationic residues of the V3 loop, in addition to conserved, positively charged residues in the CD4-induced domain on gp120. Medicinal Research Reviews DOI 10.1002/med

16


Differences were considered to be due to the elongated structure of SPL7115 and the more compact structure of SPL7013.107 Further studies suggested that the main mode of action for SPL7013 was by direct viral inactivation. X4 and R5/X4 HIV isolates with higher V3 loop charges tended to be more susceptible to SPL7013 virucidal activity by a mechanism that does not involve disruption of the viral particle or loss of gp120 from the viral surface. Inhibition of X4-HIV strains by SPL7013 occurred by irreversible or tight binding to gp120, physically blocking the binding of the virus to target cell, or by strong interactions that destroyed HIV infectivity. The mode of action against R5-HIV isolates was by strong interaction with gp120 in the gp120/CD4 complex but not gp120 alone, weakening the gp120/CD4 complex and facilitating its dissociation.108 Due to V3 loops varying in positive charge between X4 and R5 HIV-1 isolates, it is also likely that the broad potency observed for dendrimers is maintained by interactions with conserved clusters of positive charges on gp120. It is also known that V3 loops and regions surrounding V3 loops are more densely populated with positively charged residues, typical of X4-HIV isolates.107 In this regard, it is possible that the positive charges of V3 loops differ in their accessibility to the dendrimers due to the conformation of V3 loops or modifications in the N-linked glycosylation sites around V3 loops.109 Therefore, the potency against a specific virus isolate could be increased by the choice of the surface group and dendrimer size. The lack of broad-spectrum anti-HIV activity was the main weakness of SPL7013. Novel water-soluble carbosilane dendrimers that were polyphenoxo-cored or had silicon atom cores were synthesized to attain more effective and better virucidal activity.110–113 Two polyanionic carbosilane dendrimers, based on a silica atom core, were shown to inhibit HIV infection and transmission within genital mucosa and human PBMCs. G1-NS16 consists of a first-generation (generation is defined as the number of repeated layers with branching units forming the dendrimer) dendrimer fully capped on the surface with 16 naphthylsulfonate acids; G2-Sh16 is a second-generation dendrimer with 16 sulfate acid groups at the periphery.114 Both dendrimers inhibited transepithelial HIV transmission, protected the integrity of the endothelial monolayer and blocked the entry of R5 and X4-HIV isolates into PBMCs. The mode of action was associated with electrostatic interactions between functional groups of dendrimers with gp120 and other surface markers, such as CD4, CXCR4, or CCR5. According to binding of free energy for dendrimer/CD4 and dendrimer/gp120 complexes, G1-NS16 and G2-Sh16 bound strongly to gp120 at the co-receptor binding site and close to the V3 loop base.115 Another first-generation, silicon-cored carbosilane dendrimer with 16 sulfonate end-groups (G2-S16) is in advanced stages for possible application as a vaginal microbicide due to its potent and broadspectrum antiviral activity against HIV-1116 and HIV-2117 and efficacy in preventing vaginal transmission in an in vivo human BLT (bone-marrow, liver, and thymus) mouse model.118 G2-S16 acted directly on HIV, provided a barrier to infection for long periods, blocked gp120/CD4 binding, and prevented cell-to-cell transmission. Moreover, three-dimensional computer models of G2-S16 confirmed that G2-S16 formed more stable complexes with gp120 than with CD4. Curiously, G2-S16 showed high virucidal activity against R5- and X4-HIV isolates, compared to SPL7013.118 The complete inactivation of several HIV isolates likely did not involve disruption of the integrity of the viral membrane by destabilizing the core-membrane linkage or by shedding of gp120. The mode of action of G2-S16 was likely by irreversible binding to gp120, abrogating viral infectivity (Fig. 7B). A new synthetic strategy based on the use of thiol-ene chemistry was developed by the same group for the synthesis of anionic carbosilane dendrimers bearing carboxylate and sulfonate end-groups. This approach entails mild reaction conditions, few reaction steps, short reaction times, and high product yields with easy purification.111 Two second-generation polyanionic carbosilane dendrimers, which were fully capped on the surface with 16 carboxylate groups (G2-CTE16) or 16 sulfonate groups (G2-STE16), showed high inhibition of HIV in epithelial cells and PBMCs. By molecular modeling, G2-STE16 and G2-CTE16 demonstrated binding Medicinal Research Reviews DOI 10.1002/med


r 17

to CD4 and gp120, although the dendrimer/gp120 interaction showed significantly higher affinity.119 However, G2-CTE16 was less effective than G2-STE16 in all directions. G2-STE16 is larger and more flexible than G2-CTE16. Better flexibility indicates more effectiveness of the surface to interact with the cationic domains of viral receptors. The sulfonate dendrimer is larger and more flexible than the carboxylate one, with a physiological pH also forming a compound with a reduced net negative charge on the dendrimer surface. This fact is attributed to a longer spacer between the inner sulfur atoms and the end-groups and to different interactions of carboxylate and sulfonate groups with Na+ ions and water molecules. Interestingly, a carbosilane dendrimer G2-S24P with polyphenolic core and 24 sulfonate end-groups showed higher affinity to bind to CD4 than to gp120-root and to gp120 among coreceptor binding sites.115 The presence of a greater number of anionic groups at the periphery and the polyphenolic core lead to G2-S24P being less congested than dendrimers with silicon atom cores. Therefore, molecules containing polyphenolic cores have greater flexibility, and the charge density is more exposed to cellular receptors.120 Metal complexes of Co2+ , Cu2+ , Ni2+ , and Zn2+ derived from carboxylated and sulfonated PPI dendrimers with ethylenediamine cores showed high anti-HIV activity rates. These metallodendrimers had a dual behavior: reducing HIV binding to host cells in HEC-1A and VK2/E6E7 epithelial cell lines and preventing internalization of HIV into PBMCs through the blockade of membrane proteins of HIV and cells by anionic groups.121 This study showed that the antiviral properties of metallodendrimers could be modulated by the dendritic scaffold (backbone, generation, end-group) and the bound metal ions (amount, type). Recently, novel peptide dendrimers containing from 9 to 18 tryptophan residues at the periphery were shown to prevent HIV entry into target cells by binding to gp120 and gp41. Nine tryptophan residues as end-groups were sufficient to disable gp120 and gp41, although more comprehensive studies should be performed to clarify the specific mechanism of action.122

E. Dendrimers Using Other Mechanisms of Action Against HIV The mature capsid of HIV-1 is formed by the assembly of copies of a capsid protein. The protein assembly process depends on weak interprotein interactions, and the disruption of these interactions is sufficient to restrain infectivity.123 Therefore, the assembly process could be considered a strategy in searching for new antivirals that interact with the capsid protein of HIV or its domains, disrupting or altering the oligomerization capability of domains of capsid proteins. In this regard, first-generation gallic acid-triethylene glycol dendrimers with carboxylate and sulfonate end-groups were internalized into cells, likely via endocytosis or diffusion through the cellular membrane, and they inhibited the dimerization of the capsid protein of HIV-1, destabilized the quaternary structure of the C-terminal domain of the capsid protein, and impeded its assembly.124 The V3 loop plays an important role in HIV entry into cells using as its coreceptor CCR5 or CXCR4. Until now, only maraviroc has been approved by the FDA for the treatment of HIV-infected patients as a CCR5 antagonist.125 However, no clinical CXCR4 antagonists are available for the treatment of HIV-infected individuals. It is known that the high positive net charge of the V3 loop (>5) is associated with an increased preference of HIV for CXCR4.126 Thus, several viologen-based dendrimers with polycationic scaffolds have been synthesized to determine the SAR with regard to HIV inhibitory activity.127, 128 A study concluded that these compounds inhibited HIV by interactions with CXCR4, and it required an optimal number and distance of the positive charges. However, it would be ideal that novel compounds had antiviral activity against both R5- and X4-HIV isolates, as well as dual-mixed isolates. Medicinal Research Reviews DOI 10.1002/med

18


Figure 8. Schematic representation of HSV-2 envelope glycoproteins, their cell surface receptors, and the mechanism of action of dendrimers. HSV-2 contains on its surface five glycoproteins that are used mainly for its entry into target cells: gB, gC, gD, and gH-gL. Dendrimers can act in some of the steps of HSV-2 entry prevention: (i) the gB/gC-mediated initial attachment of HSV-2 to HS on host cells, (ii) interaction among gD, gD receptor, and gH-gL causing conformational changes to trigger the fusion process, and (iii) interaction with gB for complete fusion and the subsequent release of viral DNA into the cytoplasm.

7. HSV-2 ENTRY INHIBITING DENDRIMERS AS MICROBICIDES The ability of HSV-2 to efficiently infect host cells involves several receptors and pathways to facilitate virus entry into target cells. Based on the cell type, HSV-2 entry occurs by fusion with the cell membrane or by endocytosis, in which the virus triggers the fusion process with the phagocytic membrane.129 There are multiple potential targets to impede the HSV-2 entry process. We focus on this review on dendrimers that have been tested, although their exact mode of action remains unclear (Fig. 8 and Table II). Due to the high degree of complexity of the interaction of HSV glycoproteins during the entry process, it is very difficult to unequivocally identify the mode of antiviral action of many dendrimers. Most studies have been based on assumptions or realistic possibilities for accomplishing prevention of HSV-2 entry. The anti-HSV-2 activity and mechanism of action of three polyanionic PAMAM dendrimers of a fourth generation with 3,5-disulfophenylthiourea-terminated groups (SPL6039), sulfonic acid groups (SPL2784), and naphthyldisulfonic acid groups (SPL2923) were compared to those of two PLL dendrimers, the capping layers of which are 32 naphthyl disodium disulfonates (SPL2999), and 64 of which are phenyl disodium dicarbonxylates (SPL6741).130 All of the dendrimers showed high antiviral activity against HSV-2 when they were added to cells prior to the HSV-2 infection. By contrast, all of the dendrimers showed little antiviral activity when they were added to cells post-exposure to the virus, with the exception of SPL2999. These results indicated that the primary antiviral mechanism of dendrimers occurs in the first stages of HSV-2 infection, possibly blocking virus attachment to cells or interfering with adsorption and subsequent internalization. Interestingly, as occurred with SPL2923 against HIV, SPL2999 also had a secondary mechanism of action inhibiting HSV-2 in later stages of viral replication.131 Based on previous experiments and observations, this study showed that thymidine kinase was not the primary target for the anti-HSV-2 activity of SPL2999, and it speculated that SPL2999 internalized into cells and inhibited HSV-2 replication, likely by interfering with DNA synthesis. Medicinal Research Reviews DOI 10.1002/med

PAMAM PAMAM PLL

PLL

PLL

PLL

PLL

PLL

PLL

PLL

Peptide dendrimer

SPL6039 SPL2784 SPL2999

SPL6741

SPL7013

SPL7015

SPL7032

SPL7115

SB105

SB105-A10

gH625

G1

G1

G1

G2

G4

G4

G4

G5

G4 G4 G4

Generation

Amide








Ammonia Ammonia Benzhydrylamine amide

Core

18

4

4

8

32

32

32

64

24 24 32

Number of end-groups 3,5-Disulfophenylthiourea Sulfonate 1-(NHCSNH) naphthalene-3,6disulfonate 1-(NHCSNH) phenyl-3,6dicarboxylate 1-(Carboxymethoxy) naphthalene-3,6disulfonate 1-(NHC3 H4 ONH) naphthalene-3,6disulfonate 1-(NHC3 H4 ONH) naphthalene-1,3,5trisulfonate 1-(Carboxymethoxy) naphthalene-3,6disulfonate Sequence peptide chain (ASLRVRIKKQ) Sequence peptide chain (ASLRVRIKK) Sequence peptide chain (HGLASTLTRWAHYNALIRAFPrA)


gH

HSPG

HSPG

Attachment, adsorption



Attachment, adsorption, replication


Attachment, adsorption Attachment, adsorption Attachment, adsorption, replication

Targeting sites

Bis-MPA, 2,2-bis(hydroxymethyl)propionic acid; RT, reverse transcriptase; IG, integrase; GSL, glycoshpingolipid; HSPG, heparan-sulfate proteoglycan.


Code name

Table II. Characteristics of Dendrimers With Different Functional Groups As Vaginal Topical Microbicides and the Main Sites Targeting HSV-2


r 19


20


Later, three new microbicide candidates with 32 different naphthyldisulfonic acid derivatives were synthesized from the same PLL dendrimer, but the thiourea linkage was replaced by a more stable amide bond (SPL7013, SPL7015 and SPL7032).132 Three compounds had similar activities against HSV-2. However, similar to the added value of anti-HIV activity of SPL7013, it was the only dendrimer that reached full preclinical development as a topical microbicide. SPL7013 demonstrated comparable antiviral activity against HSV-2 isolates resistant to penciclovir and acyclovir, which inhibited DNA synthesis by HSV-2 DNA polymerase. Thus, the first preliminary studies on the mechanism of action of SPL7013 were performed, and dual sites of action were observed: inhibition of attachment, likely to HS; and inhibition of later stages of the HSV-2 replication with significant reduction of HSV-2 DNA synthesis in SPL7013-treated HSV-2-infected cells.133, 134 SPL7013 was selected for comparison with a dendrimer of the same family, a second-generation PLL dendrimer SPL7115. The different SARs for HIV and HSV-2 were likely a consequence of several targets involved in viral entry. The different potencies of SPL7013 and SPL7115 (SPL7013 was 140-fold better than SPL7115) revealed that the antiHSV-2 activity increased with the size of the dendrimer, suggesting that the number of anionic charges and size are important for HSV-2 inhibition. By contrast, the potency of anti-HIV activity for both dendrimers did not increase beyond the second generation. Larger dendrimers could lead to steric hindrance between viral and cell receptor interactions and could play a key role in HSV-2, compared to HIV inhibition.107 Therefore, the distinct difference in SARs requires optimizing the designs of novel compounds to enhance potency for HIV and HSV-2 as microbicides. As is the case with HIV, peptide-derived dendrimers could bind to the negatively charged sulfate and carboxyl groups of HS chains, blocking the binding of HSV-2 to cellular receptors. Screening of 15 peptide dendrimers with differences in aa sequences of their surface groups led to the selection of SB105 and its derivative SB105-A10.135 Both peptide-derived dendrimers prevented HSV-2 attachment to target cells by binding to the glycosaminoglycan moiety of cell surface HSPGs. In contrast to sulfonated dendrimers,133, 134 SB105 and SB105-A10 did not form complexes with HSV-2 envelope glycoproteins, and they also did not inhibit HSV-2 DNA synthesis. The anti-HSV-2 activity of SB105 and SB105-A10 depends on the specific aas of the peptide surface groups attached to the dendrimer scaffold. Moreover, they have advantages due to broad-spectrum antiviral activity and the low risk of emergence of drug-resistant isolates. Interestingly, the peptide dendrimer SB105-A10 was also a potent, broad-spectrum inhibitor of human papillomavirus (HPV) by binding to cellular HSPGs.136 HPV is another common STI spread through genital contact, such as vaginal and anal sex, which can be passed when the infected person has no symptoms. The synthesis of a polyamide-based azido dendrimer functionalized with a membraneinteracting peptide derived from gH of HSV-2 (gH625-644) was a candidate to inhibit HSV-2 infection.137 The gH625 peptide dendrimer showed slightly lower inhibition of HSV-2 by direct inactivation of virions. The gH625 peptide can sterically hinder the gH domain of the virus, preventing complete and functional interaction between gH and the membrane to fuse, and it can interact with other glycoproteins on the virion envelope, such as gB or gD. These systems have several advantages, such as target specificity, low toxicity, and the possibility of modifying surface characteristics easily.

8. OUTLOOK AND CONCLUSIONS Entry inhibitors play an important role in preventing HIV/HSV-2 co-infection, and they could offer new therapeutic options to treat individuals harboring multidrug-resistant viruses. Several entry inhibitors are in different stages of development as effective microbicides for the Medicinal Research Reviews DOI 10.1002/med


r 21

prevention of HIV/HSV-2 transmission. Due to the synergy between HIV and other STIs, it has been suggested that the rapid diagnosis and treatment of STIs, such as HSV-2 or HPV, could provide a reasonable and cost-effective prevention strategy against these viruses.2, 138 Polyanion-based microbicides have failed to demonstrate efficacy in clinical trials, likely due to their high polydispersity, the presence of functional end-groups randomly distributed on the surface relative to dendrimers, and the development of resistance associated with the emergence of specific mutations in gp120.54, 139 With the clinical failures of polyanions and knowing that using them as systemic agents to combat the HIV-1 infection is not a viable option, the field of microbicides is growing rapidly and has cleared the way for other classes of compounds. Several advances of this field have comprised the improvement of microbicide pharmacokinetics, better targeting, and innovative approaches, with a decreased chance for the development of resistance. In the search for these objectives, nanotechnology-based microbicides and especially dendrimers seem to be the most efficient strategy. Undoubtedly, mostly polyanionic dendrimers have very interesting features for clinical use as antivirals against HIV/HSV-2 co-infection and other STIs: they show structural uniformity and monodispersity, are biocompatible and have good biodegradability, have better biological and shelf-stability, have a greater targeting efficiency, can mimic biological receptors or cofactors by surface modifications, can entrap several drugs by encapsulation or by charge interactions, and can deliver drugs inside the cell and improve intracellular trafficking. Although most of the development of resistance occurs spontaneously due to the high mutation rate of the virus, specific aa changes can also be attributed to the selective pressure of compounds on the virus. However, if viruses overcome the inhibitory effects of dendrimers through mutations of specific aas involved in interactions with them, dendrimers can interact with other specific sites of gp120, inactivating it. Compared with other strategies against diverse STIs, dendrimers have been designed with specific functional end-groups to block both enveloped (HIV, HSV-2) and nonenveloped (HPV) viruses. Moreover, dendrimers have been designed to impede the replication of RNA viruses (HIV) and DNA viruses (HSV-2, HPV) and to interact with the envelope proteins of diverse STIs and receptors from host cells. The majority of dendrimers can target the variability of HIV/HSV-2 envelope proteins, which would explain the susceptibility of different HIV and HSV-2 variants to these entry inhibitors. A comprehensive study of the SARs of dendrimers explored the relationship between the biological activity of a dendrimer and its three dimensional structure. Molecular modeling systems can develop optimized anti-HIV/HSV-2 dendrimers. This system provides a tool for building, visualizing, analyzing, and storing models of complex molecular systems (e.g., dendrimer/virus, dendrimer/cell receptor, among others), which could increase understanding of the relationship between the structure of dendrimers and their antiviral activity. A dendrimer with dual action antiviral activity will offer ready availability, reduced cost, and advantages in preventing infections with both the HIV and HSV-2 viruses simultaneously. Moreover, the advantage of most of these dendrimers over other strategies is that they act as entry inhibitors against both viruses, blocking their replication. Currently, there are some dendrimers with dual antiviral activity, such as SB105-A10, SPL7013, and even several carbosilane dendrimers. The complexity of dendrimers is increasing because differences exist in SARs for HIV, compared with HSV-2. This complexity is more evident when dendrimers target various stages of HIV and HSV-2 entry acting by distinct mechanisms of action, especially those that also target later stages of the replication pathway. Therefore, dendrimers represent a collection of compounds that require the optimizing of their synthesis and SARs for their use as microbicides against HIV, HSV-2, and other STIs, such as HPV. All three viruses use HSPGs as initial receptors for infection, representing a common element for the development of new topical dendrimer-based microbicides that would prevent the transmission of various STIs. Medicinal Research Reviews DOI 10.1002/med

22


Two other strategies could be used: (i) a combination of a dendrimer with anti-HIV activity with another dendrimer with anti-HSV-2 activity, as well as one of them with antiviral activity against HPV; or (ii) a combination of a dendrimer with specific antiviral activity against HIV or HSV-2 (and HPV) with antivirals against HSV-2 or HIV, respectively. These strategies provide greater chances for HIV/HSV-2 protection, reducing the risk of the development of resistance, interfering at different stages of the HIV/HSV-2 lifecycles, and acting against other STIs that also fuel the HIV epidemic, such as HPV. In the case of HIV-infected subjects, R5-HIV variants are the most prevalent over the entire course of infection. These people can be treated with specific CCR5 antagonists, such as maraviroc. However, a substantial number of patients harbor dual-mix R5/X4 viruses. Pure X4-HIV isolates are rare, although they emerge in approximately 50% of infected individuals during later stages of infection. However, viruses using CXCR4 are the dominant circulating isolates in patients harboring dual-mix R5/X4 viruses.140, 141 Patients with HIV isolates that use CXCR4 for entry are not candidates for CCR5 antagonists, and they must be treated with CXCR4 antagonists, such as plerixafor. Moreover, it is important to consider R5 to X4 switching during effective highly active antiretroviral therapy and differences in the molecular evolution of these viruses between switching and nonswitching during infection.142, 143 Therefore, the great feature of dendrimers to inhibit R5, X4, and R5/X4 HIV isolates identifies these compounds as potentially broad-spectrum candidates as microbicides. Moreover, dendrimers act not only against the virus but also against cationic residues of CD4 and CCR5/CXCR4, modifying their structures and disabling them for effective interaction with the virus. Another aspect to consider is the cellular location where HIV fusion occurs24, 25 because achieving sufficient concentrations of dendrimers might be more difficult in endosomal compartments. HSV-2 has the ability to establish latency and to reactivate frequently. Therefore, a better understanding of the biological mechanisms leading HIV/HSV-2 mucosal transmission is required to identify novel generations of dendrimer-based microbicides. These microbicides directed against HIV/HSV-2 must address some requirements, including safety, efficacy, and low cost, in preventing the establishment of co-infection. To achieve all of these goals, the development of novel and optimized strategies is of outstanding relevance for the production of versatile and effective dendrimers as microbicides to prevent HIV/HSV-2 infection.

9. EXPERT OPINION The most serious contagious infections are viral in origin. There is a constant and vital need for new therapies to address the emergence of drug resistance and the side effects due to long-term treatments. While most bacterial infections have been treated with antibiotics successfully, many viral infections remain without universal treatments. Moreover, with the continuous emergence of new viral infections, the search for novel therapeutics is an endless task. In this regard, nanotechnology, and mainly dendrimers, has been demonstrated to be a potent strategy resulting in important changes at prophylactic and therapeutic levels against different viruses, such as HIV, HSV-2, HPV, or hepatitis C.144 However, there are still several challenges and obstacles that must be considered and optimized for successful translation of the microbicidal field from the laboratory to the clinical setting. Without deeper knowledge of the impact of nanoparticles on large-scale production and health, their use against diverse viruses will not be fully considered. All of these factors together other political aspects will resolve some of the outstanding issues related to dendrimers as antivirals and their clinical use. Medicinal Research Reviews DOI 10.1002/med


r 23

ACKNOWLEDGMENTS This work was (partially) funded by the RD12/0017/0037 project as part of the Plan Nacional ´ General de Evaluacion ´ y el Fondo Europeo R+D+I and co-financed by ISCIII-Subdireccion de Desarrollo Regional (FEDER), RETIC PT13/0010/0028, FIS (PI13/02016; PI14/00882), Comunidad de Madrid (S-2010/BMD-2351; S-2010/BMD-2332], CYTED 214RT0482, and ´ de la Consejer´ıa de Sanidad de la CAM to J.L.J. CIBER-BBN is Programa de Investigacion an initiative funded by the VI National R&D&i Plan2008–2011, IniciativaIngenio 2010, the Consolider Program, and CIBER Actions and financed by the ISCIII with assistance from the European Regional Development Fund. This work was supported partially by a Marie Curie International Research Staff Exchange Scheme Fellowship from the 7th European Community Framework Program, project number PIRSES-GA-2012-316730 NANOGENE, co-financed by the Polish Ministry of Science and Higher Education (grant no. W21/7PR/ 2013).

CONFLICT OF INTEREST The authors do not have commercial or other associations that might pose a conflict of interest. REFERENCES 1. Corey L, Wald A, Celum CL, Quinn TC. The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: A review of two overlapping epidemics. J Acquir Immune Defic Syndr 2004;35(5):435–445. 2. Tobian AA, Quinn TC. Herpes simplex virus type 2 and syphilis infections with HIV: an evolving synergy in transmission and prevention. Curr Opin HIV AIDS 2009;4(4):294–299. 3. Kaul R, Pettengell C, Sheth PM, Sunderji S, Biringer A, MacDonald K, Walmsley S, Rebbapragada A. The genital tract immune milieu: An important determinant of HIV susceptibility and secondary transmission. J Reprod Immunol 2008;77(1):32–40. 4. Belshe RB, Leone PA, Bernstein DI, Wald A, Levin MJ, Stapleton JT, Gorfinkel I, Morrow RL, Ewell MG, Stokes-Riner A, Dubin G, Heineman TC, Schulte JM, Deal CD. Efficacy results of a trial of a herpes simplex vaccine. N Engl J Med 2012;366(1):34–43. 5. Shin SY. Recent update in HIV vaccine development. Clin Exp Vaccine Res 2016;5(1):6–11. 6. Wang HB, Mo QH, Yang Z. HIV vaccine research: The challenge and the way forward. J Immunol Res 2015;2015:503978. 7. Pan X, Baldauf HM, Keppler OT, Fackler OT. Restrictions to HIV-1 replication in resting CD4+ T lymphocytes. Cell Res 2013;23(7):876–885. 8. Blumenthal R, Durell S, Viard M. HIV entry and envelope glycoprotein-mediated fusion. J Biol Chem 2012;287(49):40841–40849. 9. Klasse PJ. The molecular basis of HIV entry. Cell Microbiol 2012;14(8):1183–1192. 10. Wilen CB, Tilton JC, Doms RW. Molecular mechanisms of HIV entry. Adv Exp Med Biol 2012;726:223–242. 11. Cicala C, Martinelli E, McNally JP, Goode DJ, Gopaul R, Hiatt J, Jelicic K, Kottilil S, Macleod K, O’Shea A, Patel N, Van Ryk D, Wei D, Pascuccio M, Yi L, McKinnon L, Izulla P, Kimani J, Kaul R, Fauci AS, Arthos J. The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1. Proc Natl Acad Sci USA 2009;106(49):20877–20882. 12. Kondo N, Melikyan GB. Intercellular adhesion molecule 1 promotes HIV-1 attachment but not fusion to target cells. PLoS One 2012;7(9):e44827. Medicinal Research Reviews DOI 10.1002/med

24


13. Lingwood CA, Branch DR. The role of glycosphingolipids in HIV/AIDS. Discov Med 2011;11(59):303–313. 14. Moulard M, Lortat-Jacob H, Mondor I, Roca G, Wyatt R, Sodroski J, Zhao L, Olson W, Kwong PD, Sattentau QJ. Selective interactions of polyanions with basic surfaces on human immunodeficiency virus type 1 gp120. J Virol 2000;74(4):1948–1960. 15. Connell BJ, Lortat-Jacob H. Human immunodeficiency virus and heparan sulfate: From attachment to entry inhibition. Front Immunol 2013;4:385. 16. Ashish, Juncadella IJ, Garg R, Boone CD, Anguita J, Krueger JK. Conformational rearrangement within the soluble domains of the CD4 receptor is ligand-specific. J Biol Chem 2008;283(5):2761– 2772. 17. Chen B, Vogan EM, Gong H, Skehel JJ, Wiley DC, Harrison SC. Structure of an unliganded simian immunodeficiency virus gp120 core. Nature 2005;433(7028):834–841. 18. Huang CC, Tang M, Zhang MY, Majeed S, Montabana E, Stanfield RL, Dimitrov DS, Korber B, Sodroski J, Wilson IA, Wyatt R, Kwong PD. Structure of a V3-containing HIV-1 gp120 core. Science 2005;310(5750):1025–1028. 19. Huang CC, Lam SN, Acharya P, Tang M, Xiang SH, Hussan SS, Stanfield RL, Robinson J, Sodroski J, Wilson IA, Wyatt R, Bewley CA, Kwong PD. Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4. Science 2007;317(5846):1930– 1934. 20. Jensen MA, van t Wout AB. Predicting HIV-1 coreceptor usage with sequence analysis. AIDS Rev 2003;5(2):104–112. 21. Berger EA, Doms RW, Fenyo EM, Korber BT, Littman DR, Moore JP, Sattentau QJ, Schuitemaker H, Sodroski J, Weiss RA. A new classification for HIV-1. Nature 1998;391(6664):240. 22. Basmaciogullari S, Babcock GJ, Van Ryk D, Wojtowicz W, Sodroski J. Identification of conserved and variable structures in the human immunodeficiency virus gp120 glycoprotein of importance for CXCR4 binding. J Virol 2002;76(21):10791–10800. 23. Weissenhorn W, Dessen A, Harrison SC, Skehel JJ, Wiley DC. Atomic structure of the ectodomain from HIV-1 gp41. Nature 1997;387(6631):426–430. 24. Miyauchi K, Kim Y, Latinovic O, Morozov V, Melikyan GB. HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes. Cell 2009;137(3):433–444. 25. Permanyer M, Ballana E, Badia R, Pauls E, Clotet B, Este JA. Trans-infection but not infection from within endosomal compartments after cell-to-cell HIV-1 transfer to CD4+ T cells. J Biol Chem 2012;287(38):32017–32026. 26. Melikyan GB. HIV entry: A game of hide-and-fuse? Curr Opin Virol 2014;4:1–7. 27. Heldwein EE, Krummenacher C. Entry of herpesviruses into mammalian cells. Cell Mol Life Sci 2008;65(11):1653–1668. 28. Eisenberg RJ, Atanasiu D, Cairns TM, Gallagher JR, Krummenacher C, Cohen GH. Herpes virus fusion and entry: A story with many characters. Viruses 2012;4(5):800–832. 29. Spear PG. Herpes simplex virus: Receptors and ligands for cell entry. Cell Microbiol 2004;6(5):401– 410. 30. Garner JA. Herpes simplex virion entry into and intracellular transport within mammalian cells. Adv Drug Deliv Rev 2003;55(11):1497–1513. 31. Spear PG, Longnecker R. Herpesvirus entry: An update. J Virol 2003;77(19):10179–10185. 32. Spear PG, Eisenberg RJ, Cohen GH. Three classes of cell surface receptors for alphaherpesvirus entry. Virology 2000;275(1):1–8. 33. Gianni T, Amasio M, Campadelli-Fiume G. Herpes simplex virus gD forms distinct complexes with fusion executors gB and gH/gL in part through the C-terminal profusion domain. J Biol Chem 2009;284(26):17370–17382. 34. Atanasiu D, Saw WT, Cohen GH, Eisenberg RJ. Cascade of events governing cell-cell fusion induced by herpes simplex virus glycoproteins gD, gH/gL, and gB. J Virol 2010;84(23):12292–12299. Medicinal Research Reviews DOI 10.1002/med


r 25

35. Shattock RJ, Moore JP. Inhibiting sexual transmission of HIV-1 infection. Nat Rev Microbiol 2003;1(1):25–34. 36. Keller MJ, Klotman ME, Herold BC. Development of topical microbicides for prevention of human immunodeficiency virus and herpes simplex virus. Am J Reprod Immunol 2003;49(5):279–284. 37. Antimisiaris SG, Mourtas S. Recent advances on anti-HIV vaginal delivery systems development. Adv Drug Deliv Rev 2015;92:123–145. 38. Maartens G, Celum C, Lewin SR. HIV infection: Epidemiology, pathogenesis, treatment, and prevention. Lancet 2014;384(9939):258–271. 39. Shattock RJ, Rosenberg Z. Microbicides: Topical prevention against HIV. Cold Spring Harb Perspect Med 2012;2(2):a007385. 40. Gupta SK, Nutan. Clinical use of vaginal or rectally applied microbicides in patients suffering from HIV/AIDS. HIV AIDS (Auckl) 2013;5:295–307. 41. Tomalia DA. The dendritic state. Mater Today 2005;8(3):34–46. 42. Tomalia DA, Baker A, Dewald J, Hall M, Kallos G, Martin S, Roeck J, Ryder J, Smith P. A new class of polymers: Starburst-dendritic macromolecules. Polym J (Tokyo, Jpn) 1985;17:117–132. ¨ 43. Vogtle F, Richardt G, Werner N. Dendrimer Chemistry: Concepts, Syntheses, Properties, Aplications. Weinheim: Wiley-VCH; 2009. 44. Astruc D, Boisselier E, Ornelas C. Dendrimers designed for functions: from physical, photophysical, and supramolecular properties to applications in sensing, catalysis, molecular electronics, photonics, and nanomedicine. Chem Rev 2010;110(4):1857–1959. 45. Fréchet JMJ, Tomalia DA. Dendrimers and Other Dendritic Polymers. Scheirs J, editor. Chichester: Wiley; 2001. ¨ 46. Newkome GR, Moorefield CN, Vogtle F. Dendrimers and Dendrons: Concepts, Syntheses, Applications. Weinheim: Wiley-VCH Verlag; 2002. 47. Tomalia DA, Fréchet JMJ. Discovery of dendrimers and dendritic polymers: A brief historical perspective. J Polym Sci A Polym Chem 2002;40(16):2719–2728. 48. Klajnert B, Bryszewska M. Dendrimers: Properties and applications. Acta Biochim Pol 2001;48(1):199–208. 49. Abbasi E, Aval SF, Akbarzadeh A, Milani M, Nasrabadi HT, Joo SW, Hanifehpour Y, NejatiKoshki K, Pashaei-Asl R. Dendrimers: Synthesis, applications, and properties. Nanoscale Res Lett 2014;9(1):247. 50. Ina M. Dendrimer: A novel drug delivery system. J Drug Deliv Ther 2011;1(2):70–74. 51. Fasting C, Schalley CA, Weber M, Seitz O, Hecht S, Koksch B, Dernedde J, Graf C, Knapp EW, Haag R. Multivalency as a chemical organization and action principle. Angew Chem Int Ed Engl 2012;51(42):10472–10498. 52. Kiessling LL, Lamanna AC. Multivalency in biological systems. In: Schneider MP, Ed. Chemical Probes in Biology: Science at the Interface of Chemistry, Biology and Medicine, Vol. 129. Island of Spetses, Greece: NATO Science Series; 2003. p 345–357. 53. Mammen M, Choi S-K, Whitesides GM. Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew Chem Int Ed Engl 1998;37(20):2754– 2794. 54. Pirrone V, Wigdahl B, Krebs FC. The rise and fall of polyanionic inhibitors of the human immunodeficiency virus type 1. Antiviral Res 2011;90(3):168–182. 55. Rabenstein DL. Heparin and heparan sulfate: Structure and function. Nat Prod Rep 2002;19(3):312– 331. 56. Sarrazin S, Lamanna WC, Esko JD. Heparan sulfate proteoglycans. Cold Spring Harb Perspect Biol 2011;3(7):a004952. 57. Tiwari V, Maus E, Sigar IM, Ramsey KH, Shukla D. Role of heparan sulfate in sexually transmitted infections. Glycobiology 2012;22(11):1402–1412. 58. Niederhafner P, Sebestik J, Jezek J. Peptide dendrimers. J Pept Sci 2005;11(12):757–788. Medicinal Research Reviews DOI 10.1002/med

26


59. Poon GM, Gariepy J. Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells. Biochem Soc Trans 2007;35(Pt 4):788–793. 60. Bon I, Lembo D, Rusnati M, Clo A, Morini S, Miserocchi A, Bugatti A, Grigolon S, Musumeci G, Landolfo S, Re MC, Gibellini D. Peptide-derivatized SB105-A10 dendrimer inhibits the infectivity of R5 and X4 HIV-1 strains in primary PBMCs and cervicovaginal histocultures. PLoS One 2013;8(10):e76482. 61. Bishop JR, Schuksz M, Esko JD. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 2007;446(7139):1030–1037. 62. Castellano LM, Shorter J. The surprising role of amyloid fibrils in HIV infection. Biology (Basel) 2012;1(1):58–80. 63. Svajger U, Anderluh M, Jeras M, Obermajer N. C-type lectin DC-SIGN: An adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity. Cell Signal 2010;22(10):1397– 1405. 64. van Kooyk Y, Geijtenbeek TB. DC-SIGN: Escape mechanism for pathogens. Nat Rev Immunol 2003;3(9):697–709. 65. Anderluh M, Jug G, Svajger U, Obermajer N. DC-SIGN antagonists, a potential new class of anti-infectives. Curr Med Chem 2012;19(7):992–1007. 66. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS, KewalRamani VN, Littman DR, Figdor CG, van Kooyk Y. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 2000;100(5):587– 597. 67. Geijtenbeek TB, van Kooyk Y. DC-SIGN: A novel HIV receptor on DCs that mediates HIV-1 transmission. Curr Top Microbiol Immunol 2003;276:31–54. 68. Neumann AK, Thompson NL, Jacobson K. Distribution and lateral mobility of DC-SIGN on immature dendritic cells – implications for pathogen uptake. J Cell Sci 2008;121(Pt 5):634–643. 69. Wu L, KewalRamani VN. Dendritic-cell interactions with HIV: Infection and viral dissemination. Nat Rev Immunol 2006;6(11):859–868. 70. Feinberg H, Castelli R, Drickamer K, Seeberger PH, Weis WI. Multiple modes of binding enhance the affinity of DC-SIGN for high mannose N-linked glycans found on viral glycoproteins. J Biol Chem 2007;282(6):4202–4209. 71. Ji X, Chen Y, Faro J, Gewurz H, Bremer J, Spear GT. Interaction of human immunodeficiency virus (HIV) glycans with lectins of the human immune system. Curr Protein Pept Sci 2006;7(4):317– 324. 72. Rojo J, Delgado R. Glycodendritic structures: Promising new antiviral drugs. J Antimicrob Chemother 2004;54(3):579–581. 73. Tsegaye TS, Pohlmann S. The multiple facets of HIV attachment to dendritic cell lectins. Cell Microbiol 2010;12(11):1553–1561. 74. Turville S, Wilkinson J, Cameron P, Dable J, Cunningham AL. The role of dendritic cell C-type lectin receptors in HIV pathogenesis. J Leukoc Biol 2003;74(5):710–718. 75. Wang SK, Liang PH, Astronomo RD, Hsu TL, Hsieh SL, Burton DR, Wong CH. Targeting the carbohydrates on HIV-1: Interaction of oligomannose dendrons with human monoclonal antibody 2G12 and DC-SIGN. Proc Natl Acad Sci USA 2008;105(10):3690–3695. 76. Zagar E, Zigon M. Aliphatic hyperbranched polyesters based on 2, 2-bis(methylol)propionic acid— Determination of structure, solution and bulk properties. Prog Polym Sci 2011;36(1):53–88. 77. Tabarani G, Reina JJ, Ebel C, Vives C, Lortat-Jacob H, Rojo J, Fieschi F. Mannose hyperbranched dendritic polymers interact with clustered organization of DC-SIGN and inhibit gp120 binding. FEBS Lett 2006;580(10):2402–2408. 78. Reina JJ, Sattin S, Invernizzi D, Mari S, Martinez-Prats L, Tabarani G, Fieschi F, Delgado R, Nieto PM, Rojo J, Bernardi A. 1,2-Mannobioside mimic: Synthesis, DC-SIGN interaction by NMR and docking, and antiviral activity. ChemMedChem 2007;2(7):1030–1036.



r 27

79. Sattin S, Daghetti A, Thepaut M, Berzi A, Sanchez-Navarro M, Tabarani G, Rojo J, Fieschi F, Clerici M, Bernardi A. Inhibition of DC-SIGN-mediated HIV infection by a linear trimannoside mimic in a tetravalent presentation. ACS Chem Biol 2010;5(3):301–312. 80. Varga N, Sutkeviciute I, Ribeiro-Viana R, Berzi A, Ramdasi R, Daghetti A, Vettoretti G, Amara A, Clerici M, Rojo J, Fieschi F, Bernardi A. A multivalent inhibitor of the DC-SIGN dependent uptake of HIV-1 and dengue virus. Biomaterials 2014;35(13):4175–4184. 81. Garcia-Vallejo JJ, Koning N, Ambrosini M, Kalay H, Vuist I, Sarrami-Forooshani R, Geijtenbeek TB, van Kooyk Y. Glycodendrimers prevent HIV transmission via DC-SIGN on dendritic cells. Int Immunol 2013;25(4):221–233. 82. Dam TK, Brewer CF. Lectins as pattern recognition molecules: The effects of epitope density in innate immunity. Glycobiology 2010;20(3):270–279. 83. Fantini J, Hammache D, Pieroni G, Yahi N. Role of glycosphingolipid microdomains in CD4dependent HIV-1 fusion. Glycoconj J 2000;17(3 -4):199–204. 84. Lingwood CA. Glycosphingolipid functions. Cold Spring Harb Perspect Biol 2011;3(7):a004788. 85. Schneider-Schaulies J, Schneider-Schaulies S. Viral infections and sphingolipids. Handb Exp Pharmacol 2013(216):321–340. 86. Viard M, Parolini I, Rawat SS, Fecchi K, Sargiacomo M, Puri A, Blumenthal R. The role of glycosphingolipids in HIV signaling, entry and pathogenesis. Glycoconj J 2004;20(3):213–222. 87. Wilflingseder D, Stoiber H. Float on: Lipid rafts in the lifecycle of HIV. Front Biosci 2007;12:2124– 2135. 88. Kensinger RD, Yowler BC, Benesi AJ, Schengrund CL. Synthesis of novel, multivalent glycodendrimers as ligands for HIV-1 gp120. Bioconjug Chem 2004;15(2):349–358. 89. Kensinger RD, Catalone BJ, Krebs FC, Wigdahl B, Schengrund CL. Novel polysulfated galactosederivatized dendrimers as binding antagonists of human immunodeficiency virus type 1 infection. Antimicrob Agents Chemother 2004;48(5):1614–1623. 90. Morales-Serna JA, Boutureira O, Serra A, Matheu MI, Diaz Y, Castillon S. Synthesis of hyperbranched β-galceramide-containing dendritic polymers that bind HIV-1 rgp120. Eur J Org Chem 2010;2010(14):2657–2660. 91. Perez-Anes A, Stefaniu C, Moog C, Majoral JP, Blanzat M, Turrin CO, Caminade AM, Rico-Lattes I. Multivalent catanionic GalCer analogs derived from first generation dendrimeric phosphonic acids. Bioorg Med Chem 2010;18(1):242–248. 92. Perez-Anes A, Spataro G, Coppel Y, Moog C, Blanzat M, Turrin CO, Caminade AM, Rico-Lattes I, Majoral JP. Phosphonate terminated PPH dendrimers: influence of pendant alkyl chains on the in vitro anti-HIV-1 properties. Org Biomol Chem 2009;7(17):3491–3498. 93. Han S, Yoshida D, Kanamoto T, Nakashima H, Uryu T, Yoshida T. Sulfated oligosaccharide cluster with polylysine core scaffold as a new anti-HIV dendrimer. Carbohydr Polym 2010;80(4):1111–1115. 94. Han S, Kanamoto T, Nakashima H, Yoshida T. Synthesis of a new amphiphilic glycodendrimer with antiviral functionality. Carbohydr Polym 2012;90(2):1061–1068. 95. Rosa Borges A, Wieczorek L, Johnson B, Benesi AJ, Brown BK, Kensinger RD, Krebs FC, Wigdahl B, Blumenthal R, Puri A, McCutchan FE, Birx DL, Polonis VR, Schengrund CL. Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1. Virology 2010;408(1):80–88. 96. Schneider-Schaulies J, Schneider-Schaulies S. Sphingolipids in viral infection. Biol Chem 2015;396(6-7):585–595. 97. Clayton R, Hardman J, LaBranche CC, McReynolds KD. Evaluation of the synthesis of sialic acid-PAMAM glycodendrimers without the use of sugar protecting groups, and the anti-HIV-1 properties of these compounds. Bioconjug Chem 2011;22(10):2186–2197. 98. Hug P, Lin HM, Korte T, Xiao X, Dimitrov DS, Wang JM, Puri A, Blumenthal R. Glycosphingolipids promote entry of a broad range of human immunodeficiency virus type 1 isolates into cell lines expressing CD4, CXCR4, and/or CCR5. J Virol 2000;74(14):6377–6385.


28


99. Mahfoud R, Mylvaganam M, Lingwood CA, Fantini J. A novel soluble analog of the HIV-1 fusion cofactor, globotriaosylceramide (Gb(3)), eliminates the cholesterol requirement for high affinity gp120/Gb(3) interaction. J Lipid Res 2002;43(10):1670–1679. 100. Manes S, del Real G, Lacalle RA, Lucas P, Gomez-Mouton C, Sanchez-Palomino S, Delgado R, Alcami J, Mira E, Martinez AC. Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection. EMBO Rep 2000;1(2):190–196. 101. Puryear WB, Gummuluru S. Role of glycosphingolipids in dendritic cell-mediated HIV-1 transinfection. Adv Exp Med Biol 2013;762:131–153. 102. Rizzuto CD, Wyatt R, Hernandez-Ramos N, Sun Y, Kwong PD, Hendrickson WA, Sodroski J. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 1998;280(5371):1949–1953. 103. Marin MH, Pena LP, Tanty CR, Higginson Clarke D, Arenas MA, Noguerol KR, Leon CS. Antigenic activity of three chimeric synthetic peptides of the transmembrane (gp41) and the envelope (gp120) glycoproteins of HIV-1 virus. Prep Biochem Biotechnol 2004;34(3):227–237. 104. Witvrouw M, Fikkert V, Pluymers W, Matthews B, Mardel K, Schols D, Raff J, Debyser Z, De Clercq E, Holan G, Pannecouque C. Polyanionic (i.e., polysulfonate) dendrimers can inhibit the replication of human immunodeficiency virus by interfering with both virus adsorption and later steps (reverse transcriptase/integrase) in the virus replicative cycle. Mol Pharmacol 2000;58(5):1100– 1108. 105. Nandy B, Bindu DH, Dixit NM, Maiti PK. Simulations reveal that the HIV-1 gp120-CD4 complex dissociates via complex pathways and is a potential target of the polyamidoamine (PAMAM) dendrimer. J Chem Phys 2013;139(2):024905. 106. McCarthy TD, Karellas P, Henderson SA, Giannis M, O’Keefe DF, Heery G, Paull JR, Matthews BR, Holan G. Dendrimers as drugs: discovery and preclinical and clinical development of dendrimer-based microbicides for HIV and STI prevention. Mol Pharm 2005;2(4):312–318. 107. Tyssen D, Henderson SA, Johnson A, Sterjovski J, Moore K, La J, Zanin M, Sonza S, Karellas P, Giannis MP, Krippner G, Wesselingh S, McCarthy T, Gorry PR, Ramsland PA, Cone R, Paull JR, Lewis GR, Tachedjian G. Structure activity relationship of dendrimer microbicides with dual action antiviral activity. PLoS One 2010;5(8):e12309. 108. Nandy B, Saurabh S, Sahoo AK, Dixit NM, Maiti PK. The SPL7013 dendrimer destabilizes the HIV-1 gp120-CD4 complex. Nanoscale 2015;7(44):18628–18641. 109. Sterjovski J, Roche M, Churchill MJ, Ellett A, Farrugia W, Gray LR, Cowley D, Poumbourios P, Lee B, Wesselingh SL, Cunningham AL, Ramsland PA, Gorry PR. An altered and more efficient mechanism of CCR5 engagement contributes to macrophage tropism of CCR5-using HIV-1 envelopes. Virology 2010;404(2):269–278. 110. Arnáiz E, Vacas Cordoba E, Galan M, Pion M, Gomez R, Munoz-Fernandez MA, de la Mata FJ. Synthesis of anionic carbosilane dendrimers via “click chemistry” and their antiviral properties against HIV. J Polym Sci A Polym Chem 2014;52(8):1099–1112. 111. Galan M, Sanchez Rodriguez J, Jimenez JL, Relloso M, Maly M, de la Mata FJ, Munoz-Fernandez MA, Gomez R. Synthesis of new anionic carbosilane dendrimers via thiol-ene chemistry and their antiviral behaviour. Org Biomol Chem 2014;12(20):3222–3237. 112. Galan M, Sanchez-Rodriguez J, Cangiotti M, Garcia-Gallego S, Jimenez JL, Gomez R, Ottaviani MF, Munoz-Fernandez MA, de la Mata FJ. Antiviral properties against HIV of water soluble copper carbosilane dendrimers and their EPR characterization. Curr Med Chem 2012;19(29):4984–4994. 113. Rasines B, Sanchez-Nieves J, Maiolo M, Maly M, Chonco L, Jimenez JL, Munoz-Fernandez MA, de la Mata FJ, Gomez R. Synthesis, structure and molecular modelling of anionic carbosilane dendrimers. Dalton Trans 2012;41(41):12733–12748. 114. Vacas Cordoba E, Arnaiz E, Relloso M, Sanchez-Torres C, Garcia F, Perez-Alvarez L, Gomez R, de la Mata FJ, Pion M, Munoz-Fernandez MA. Development of sulphated and naphthylsulphonated carbosilane dendrimers as topical microbicides to prevent HIV-1 sexual transmission. AIDS 2013;27(8):1219–1229. Medicinal Research Reviews DOI 10.1002/med


r 29

115. Sepulveda-Crespo D, Gomez R, De La Mata FJ, Jimenez JL, Munoz-Fernandez MA. Polyanionic carbosilane dendrimer-conjugated antiviral drugs as efficient microbicides: Recent trends and developments in HIV treatment/therapy. Nanomedicine 2015;11(6):1481–1498. 116. Chonco L, Pion M, Vacas E, Rasines B, Maly M, Serramia MJ, Lopez-Fernandez L, De la Mata J, Alvarez S, Gomez R, Munoz-Fernandez MA. Carbosilane dendrimer nanotechnology outlines of the broad HIV blocker profile. J Control Release 2012;161(3):949–958. 117. Briz V, Sepulveda-Crespo D, Diniz AR, Borrego P, Rodes B, de la Mata FJ, Gomez R, Taveira N, Munoz-Fernandez MA. Development of water-soluble polyanionic carbosilane dendrimers as novel and highly potent topical anti-HIV-2 microbicides. Nanoscale 2015;7(35):14669–14683. 118. Sepulveda-Crespo D, Serramia MJ, Tager AM, Vrbanac V, Gomez R, De La Mata FJ, Jimenez JL, Munoz-Fernandez MA. Prevention vaginally of HIV-1 transmission in humanized BLT mice and mode of antiviral action of polyanionic carbosilane dendrimer G2-S16. Nanomedicine 2015;11(6):1299–1308. ´ ˜ 119. Sánchez-Rodr´ıguez J, D´ıaz L, Galán M, Maly M, Gomez R, de la Mata FJ, Jiménez JL, MunozFernández MA. Anti-human immunodeficiency virus activity of thiol-ene carbosilane dendrimers and their potential development as a topical microbicide. J Biomed Nanotechnol 2015;11(10):1783– 1798. ˜ 120. Sánchez-Nieves J, Ortega P, Munoz-Fernandez MA, Gomez R, De la Mata FJ. Synthesis of carbosilane dendrons and dendrimers derived from 1,3,5-trihydroxybenzene. Tetrahedron 2010;66(47):9203–9213. 121. Garcia-Gallego S, Diaz L, Jimenez JL, Gomez R, de la Mata FJ, Munoz-Fernandez MA. HIV-1 antiviral behavior of anionic PPI metallo-dendrimers with EDA core. Eur J Med Chem 2015;98:139– 148. 122. Rivero-Buceta E, Doyaguez EG, Colomer I, Quesada E, Mathys L, Noppen S, Liekens S, Camarasa MJ, Perez-Perez MJ, Balzarini J, San-Felix A. Tryptophan dendrimers that inhibit HIV replication, prevent virus entry and bind to the HIV envelope glycoproteins gp120 and gp41. Eur J Med Chem 2015;106:34–43. 123. Neira JL. The capsid protein of human immunodeficiency virus: designing inhibitors of capsid assembly. FEBS J 2009;276(21):6110–6117. 124. Domenech R, Abian O, Bocanegra R, Correa J, Sousa-Herves A, Riguera R, Mateu MG, Fernandez-Megia E, Velazquez-Campoy A, Neira JL. Dendrimers as potential inhibitors of the dimerization of the capsid protein of HIV-1. Biomacromolecules 2010;11(8):2069–2078. 125. Lieberman-Blum SS, Fung HB, Bandres JC. Maraviroc: A CCR5-receptor antagonist for the treatment of HIV-1 infection. Clin Ther 2008;30(7):1228–1250. 126. Lopez de Victoria A, Kieslich CA, Rizos AK, Krambovitis E, Morikis D. Clustering of HIV-1 subtypes based on gp120 V3 loop electrostatic properties. BMC Biophys 2012;5:3. 127. Asaftei S, Huskens D, Schols D. HIV-1 X4 activities of polycationic "viologen" based dendrimers by interaction with the chemokine receptor CXCR4: Study of structure-activity relationship. J Med Chem 2012;55(23):10405–10413. 128. Asaftei S, De Clercq E. "Viologen" dendrimers as antiviral agents: the effect of charge number and distance. J Med Chem 2010;53(9):3480–3488. 129. Karasneh GA, Shukla D. Herpes simplex virus infects most cell types in vitro: Clues to its success. Virol J 2011;8:481. 130. Bourne N, Stanberry LR, Kern ER, Holan G, Matthews B, Bernstein DI. Dendrimers, a new class of candidate topical microbicides with activity against herpes simplex virus infection. Antimicrob Agents Chemother 2000;44(9):2471–2474. 131. Gong Y, Matthews B, Cheung D, Tam T, Gadawski I, Leung D, Holan G, Raff J, Sacks S. Evidence of dual sites of action of dendrimers: SPL-2999 inhibits both virus entry and late stages of herpes simplex virus replication. Antiviral Res 2002;55(2):319–329. 132. Bernstein DI, Stanberry LR, Sacks S, Ayisi NK, Gong YH, Ireland J, Mumper RJ, Holan G, Matthews B, McCarthy T, Bourne N. Evaluations of unformulated and formulated Medicinal Research Reviews DOI 10.1002/med

30

133. 134.

135.

136.

137.

138. 139.

140.

141.

142.

143.

144.

´ r SEPULVEDA-CRESPO ET AL. dendrimer-based microbicide candidates in mouse and guinea pig models of genital herpes. Antimicrob Agents Chemother 2003;47(12):3784–3788. Rupp R, Rosenthal SL, Stanberry LR. VivaGel (SPL7013 Gel): A candidate dendrimer–microbicide for the prevention of HIV and HSV infection. Int J Nanomedicine 2007;2(4):561–566. Gong E, Matthews B, McCarthy T, Chu J, Holan G, Raff J, Sacks S. Evaluation of dendrimer SPL7013, a lead microbicide candidate against herpes simplex viruses. Antiviral Res 2005;68(3):139– 146. Luganini A, Nicoletto SF, Pizzuto L, Pirri G, Giuliani A, Landolfo S, Gribaudo G. Inhibition of herpes simplex virus type 1 and type 2 infections by peptide-derivatized dendrimers. Antimicrob Agents Chemother 2011;55(7):3231–3239. Donalisio M, Rusnati M, Civra A, Bugatti A, Allemand D, Pirri G, Giuliani A, Landolfo S, Lembo D. Identification of a dendrimeric heparan sulfate-binding peptide that inhibits infectivity of genital types of human papillomaviruses. Antimicrob Agents Chemother 2010;54(10):4290–4299. Tarallo R, Carberry TP, Falanga A, Vitiello M, Galdiero S, Galdiero M, Weck M. Dendrimers functionalized with membrane-interacting peptides for viral inhibition. Int J Nanomedicine 2013;8:521– 534. Corey L. Synergistic copathogens – HIV-1 and HSV-2. N Engl J Med 2007;356(8):854–856. Este JA, Schols D, De Vreese K, Van Laethem K, Vandamme AM, Desmyter J, De Clercq E. Development of resistance of human immunodeficiency virus type 1 to dextran sulfate associated with the emergence of specific mutations in the envelope gp120 glycoprotein. Mol Pharmacol 1997;52(1):98–104. Moyle GJ, Wildfire A, Mandalia S, Mayer H, Goodrich J, Whitcomb J, Gazzard BG. Epidemiology and predictive factors for chemokine receptor use in HIV-1 infection. J Infect Dis 2005;191(6):866– 872. Huang W, Toma J, Stawiski E, Fransen S, Wrin T, Parkin N, Whitcomb JM, Coakley E, Hecht FM, Deeks SG, Gandhi RT, Eshleman SH, Petropoulos CJ. Characterization of human immunodeficiency virus type 1 populations containing CXCR4-using variants from recently infected individuals. AIDS Res Hum Retroviruses 2009;25(8):795–802. Delobel P, Sandres-Saune K, Cazabat M, Pasquier C, Marchou B, Massip P, Izopet J. R5 to X4 switch of the predominant HIV-1 population in cellular reservoirs during effective highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2005;38(4):382–392. Mild M, Kvist A, Esbjornsson J, Karlsson I, Fenyo EM, Medstrand P. Differences in molecular evolution between switch (R5 to R5X4/X4-tropic) and non-switch (R5-tropic only) HIV-1 populations during infection. Infect Genet Evol 2010;10(3):356–364. Szunerits S, Barras A, Khanal M, Pagneux Q, Boukherroub R. Nanostructures for the inhibition of viral infections. Molecules 2015;20(8):14051–14081.

Daniel Sepúlveda-Crespo earned a Ph.D. in biochemistry, molecular biology, biomedicine, and biotechnology (Molecular Biosciences). Daniel obtained his degree in chemical engineering at the University Complutense of Madrid in 2010. He obtained his Ph.D. with International Mention from University Autónoma (Madrid, Spain) in 2016 under the supervision of Prof. Ma Angeles Muñoz-Fernandez and Prof. José Luis Jimenez. During his predoctoral fellowship at Laboratorio de InmunoBiologia Molecular and Plataforma de Laboratorio in Hospital General Universitario Gregorio Maraño´ n, he worked on the development of novel nanocompounds as topical microbicides with activity against HIV. His main field of interest is the study of the chemistry of dendrimers and diverse strategies of combination with the aim of finding new preventive targets against HIV and Medicinal Research Reviews DOI 10.1002/med


r 31

other sexually transmitted infections. His work has been published in six peer-reviewed journals over 4 years. Rafael Ceña-Diez is currently a Ph.D. student at Laboratorio de InmunoBiologia Molecular and Plataforma de Laboratorio in Hospital General Universitario Gregorio Maraño´ n. Rafael obtained his degree in biology and biochemistry at the University of Navarra in 2013. During his predoctoral fellowship, he worked with nanocompounds on the development of novel topical microbicides with dual activity against HIV and HSV-2, in vitro and in vivo. José Luis Jiménez obtained his Ph.D. in biology from Complutense University (Madrid, Spain) ´ in 2002 under the supervision of Prof. Ma Angeles Muñoz-Fernández and Prof. Manuel Fresno, on research into PDE IV inhibitors and HIV replication. Since then and currently, he works at the Gregorio Marañon Hospital (IiSGM) searching for alternative therapies against HIV (therapeutic and prophylactic), using nanomedicine and more specifically dendrimers. In the last 6 years, he has published more than 30 articles (1st quartile [H index: 14)], has participated in five national projects as principal investigator, obtained three patents, and was supervisor of two Ph.D. thesis, and he also has experience in advising Master’s degree students. ´ Ma Angeles Muñoz-Fernández (Ph.D., M.D.) earned a Ph.D. in biology and medicine, and she is specialist in immunology. She has been involved in HIV infection and other virological diseases research since 1992 working in the "Hospital General Universitario Gregorio Maraño´ n" and in collaboration with other European and American researchers, such as with Prof. T. Merigan of Stanford University. She is one of the leaders of the Spanish Network for HIV Research and is director of the Spanish HIV HGM BioBank. Her laboratory has been actively involved in the dendrimer field since 2003. In the last 6 years, she has published 111 articles (1st decile), participated in 13 International research projects as coordinator or IP and has obtained 14 patents (H index 33).
