Essay: US28 HCMV (Human Cyto-Megalo Virus) encoded vGPCR

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US28 is one of four HCMV (Human Cyto-Megalo Virus) encoded vGPCRs that has been extensively studied over the years. It exhibits high constitutive activity and posseses the ability to bind ligands from two separate chemokine classes. The subsequent distinct signalling pathways are initated upon G-protein activation and interestingly, dependent on the type (or the absence) of bound ligand. The functional consequences of US28 and the signalling pathways have been proposed to mediate various diseases such as cardiovascular diseases and tumorigenesis. US28 has also been proposed to act as a co-receptor for HIV-1 (Human Immunodeficiency Virus) entry in CD4+ coexpressing cells. US28 thus becomes an important pharmacological target in combating HCMV associated diseases. This review will focus on the general binding, signalling and regulation of US28, their functional consequences in mediating diseases and most importantly, the potential of using small molecules and FTP (Fusion-Toxicity-Proteins) in ameliorating HCMV-associated diseases and preventing life-threatening states for immunocompromised individuals. The underlying articles for this review has been gathered either by using MeSH (Medical Subject Headings) search terms or direct search terms in PubMed on NCBI’s (National Center for Biotechnology Information) website.
 
Introduction

HCMV is a fairly common β-virus that lies latent throughout the life of the typical, healthy human being [1]. Normally, clinical manifestations are not present unless the infected individual is immunocompromised such as in AIDS-, cancer-, and transplant- patients and in neonates, whom as a result can end up in severe or even fatal states [2]. Furthermore, HCMV has been linked to the development of cardiovascular diseases, for instance atherosclerosis, restenosis after angioplasty, and vascular sclerosis in transplants (“associated with chronic rejection of transplanted solid organs”) [1]. An association between HCMV and tumorigenesis and HIV-progression has also been clearly established [3, 4].

HCMV encodes four vGPCRs, US27, US28, UL33 and UL78 [5]. All of these vGPCRs show high homology to chemokine receptors existent endogenously in mammalians, and subsequently, they bind to chemokines and interfere with chemokine signalling and thus conflict with the host’s chemokine and immune system. Among the four vGPCRs, US28 is the most extensively studied due to its high ligand, independent constitutive activation of phospholipase C and NF-kB as well as its ability to bind both CC-chemokines (e.g. RANTES and MCP-1 ) and CX3C-chemokines (Fractalkine) [5].

The functional consequence of both of these characteristics, high constitutive activity and binding of different chemokines, are factors that are involved in the development of the various HCMV-associated diseases [1, 5]. Lastly, US28 acts as a chemokine scavenger receptor through its ability to undergo constitutive internalization, which combined with its constitutive regulation of signalling, ultimately augments viral dissemination [6, 7].

In light of the functional, pathophysiological significance of US28, it’s cognate signalling pathways and regulation/internalization, targeting US28 with small molecule variants of VUF2774 as well as the FTP-variant, F49A-FTP, may have a profound impact in treating HCMV and thus in preventing life-threatening, HCMV-associated diseases in immunocompromised patients.

Material & Method

The articles used in the writing of this review has mainly been found using MeSH search terms, which is an “NLM [National Library of Medicine] controlled vocabulary thesaurus used for indexing articles for PubMed”[8] on NCBI’s website. Since US28 is a fairly narrow topic, a search using MeSH merely yielded a total of 82 articles. After having completed the synopsis in guidance with the bachelor supervisor combined with reading some reviews to gather background knowledge, the articles were found by taking one topic at a time. The articles were then picked after relevance, article type (mainly original articles) and publishing year (after the year 2000):

Generally about US28:

To acquire a general understanding of US28, reviews were the first articles that were searched for.

Using MeSH search terms “US28” and filtering with “reviews” (US28 receptor, Cytomegalovirus”[Supplementary Concept] AND Review[ptyp]), 7 articles were found. The article “Human Cytomegalovirus US28: a functionally selective chemokine binding receptor” was selected because of relevance (favoured words such as: “Human Cytomegalovirus”, “US28”, “chemokine”, “Binding”, “Receptor”.) as well as not being too specific. The publishing year was 2001, and thus after 2000.

To find another general review I searched directly on PubMed’s database, which yielded 20 articles with the same search profile as above. “US28, a Virally-Encoded GPCR as an Antiviral Target for Human Cytomegalovirus Infection.” was selected again because of its general article title, relevance (“US28”, “Human cytomegalovirus”, “Antiviral”) and lastly the recent year of publishing, 2017.

Upon reading the reviews, a greater, general understanding of US28 was acquired. This allowed for a more specific search profile for the following topics:

Ligand binding and signalling

• MeSH search terms: “US28 receptor “, “Protein binding”, “GTP-protein binding”, “Type C phospholipases”, “signal transduction”.

• Number of hits: 5-10 articles

• Favoured words: “binding”, “Chemokine receptor”, “signalling”, “constitutive activity”, “phospholipase”

• Additional:

o General mechanism of binding and signalling was favoured.

Regulation and internalization/recycling

• MeSH search terms/ filter: “US28 receptor “, “Beta arrestins”, “Internalization”.

• Number of hits: 3 articles

• Favoured words: “Internalization, Beta arrestins” and “regulation”.

• Additional:

o General mechanism of regulation and internalization was favoured.

Pathophysiological relevance of US28

Cardiovascular disease

• MeSH search terms: “US28 receptor and “Smooth muscle”.

• Number of hits: 6 articles

• Favoured words: “Migration/motility”, “chemokine receptor”.

• Additional

o Since the search term “Cardiovascular disease” in combination yielded 0 original articles, the term “smooth muscle” was used instead; an underlying factor for US28-mediated cardiovascular disease.

o Since “migration/motility” appeared twice out of the 6 hits, the most recent article with the most recent publishing year was chosen (2009)

Oncogenesis

• MeSH search terms”US28 receptor and “carcinogenesis”/”Neoplasms”

• Number of hits: 4-10

• Favoured words: “Tumor/Tumorigenesis, “Cancer”, “angiogenesis”, “proliferation”

• Additional

o The more general mechanisms of tumorigenesis were used as criteria, thereby filtering away the articles that were more specific to a certain type of cancer (However, colorectal cancer article [9] was picked as an example due to its incidence rate as well as publishing year [2016]).

US28 as pharmacological target

Small molecules:

• MeSH search terms: “US28 receptor”, “small molecules”, “drug inverse agonism”, “drug design”,

• Number of hits: 1-10

• Favoured words: “Inverse agonism”, “Small molecules”, “modulate”

• Additional

o Articles about VUF2774 and VUF2774 variants were favoured.

FTP (Fusion Toxicity Protein)

• PubMed search terms: “US28 toxin protein”

• Number of hits: 5 hits

• Favoured words: “Toxin protein”

• Additional:

o Searching directly in PubMed was carried because there were either no MeSH terms for FTP/ toxin protein or no hits appeared. This was no problem (in terms of too many hits) since targeting US28 with FTP is a relatively new principle.

o The most recent articles were used (2015-2017).

Lastly, it’s important to note that upon reading the articles (categorized under the specific topics), some of the respective articles were also relevant under the various other topics.

Results and Discussion

Ligand binding

The HCMV encoded US28 chemokine GPCR exhibits high similarity with chemokine receptors CCR1, CCR5, CX3CR1 and thus has affinity for chemokines such as CCL5 (RANTES), CCL2 (MCP-1), and CX3CL1 (Fractalkine) [10]. High affinity binding of chemokines to US28 has shown to be highly dependent on a 6 amino acid long sequence located in the N-Terminus of the receptor [1]. However, the specific high affinity binding of the aforementioned
ligands “require different residues in this hexapeptide region “[1]. The CX3CL1 will be used to describe the general mechanism of ligand binding to US28 receptor.

The binding of CX3CL1 (Fractalkine) to US28 receptor follows a two-site binding model [11]. The external and internal interaction between ligand and receptor make up site 1 and 2 respectively. The latter involves Fractalkine’s N-terminus’ projection into the helical core of US28, extending to the base of the extracellular cavity, utilizing a vital binding region for small molecules to many GPCRs [11]. Site 1 makes up the external binding site, comprising the interaction between the chemokine globular body of Fractalkine with the N-terminus and extracellular loops of US28. More specifically, the primary interaction occurs between a groove created by the “junction between the β sheet and the N loop” on the surface of Fractalkine and the N-terminus of US28 receptor [11]. Site 1 accounts for the largest binding area and consists mostly of van der Waals interactions and to a lesser degree of hydrogen bonds (44 and 13 accordingly).

US28 Signalling

US28 chemokine receptor activates a diverse series of G-proteins, which leads to a subsequent diverse series of signalling pathways. Activation of G-proteins via US28 happens upon ligand binding as well as in its mere unbound state, which leads to higher constitutive activity in US28 expressing cells [10]. The increased constitutive agonist independent activity through viral GPCR, in this case US28, is a typical strategy whereupon viruses intensify signalling, resulting in increased survival and dissemination of the virus and thereby furthering viral pathogenesis [11].

PLC-β activation and PLC-β dependent pathways have been shown to occur in all US28 expressing cells in all cell types through agonist independent activation [10]. For example, in COS-7 cells, transient expression of US28 leads to increased constitutive activation of PLC through Gαq/11 subunit of Gq/11 [12]. The DRY motif situated at the end of TM-3 domain is critical for US28 signalling to PLC-β [1]. Furthermore, Gq/11 also stimulates NF-κB; a transcription factor involved in immune response and inflammatory events and thus possesses pathophysiological importance [12]. Stimulation of NF-κB via Gq/11 occurs through Gαq/11 subunit and βγ subunit, specifically the β2γ1 dimer [12]. Lastly, the constitutive activity of US28 also stimulates Gαi/o, which leads to inhibition of adenylate cyclase and subsequent inhibition of PKA [12].

US28 has affinity for chemokines such as RANTES, MCP-1, and Fractalkine, all of which, upon binding, initiate various different intracellular changes such as increased intracellular calcium levels, small G-protein Rho activation and focal adhesion kinase (FAK) activation [10]. CX3CL1 is special, because it displays both agonist and inverse agonistic features upon interacting with US28 via Gq/11. When CX3CL1 is bound to US28 in US28 expressing cells, a reduction in Gq/11 mediated inositol phosphate (~35% inhibition [5]) and NF-κB is seen [12]. In signalling assays, CX3CL1 bound to US28 has shown to initially exhibit agonistic signalling but subsequently leads to overall reduced signalling due to “ligand induced internalization and degradation” [11]. For this reason CX3CL1 cannot be seen as an inverse agonist, since it has no effect on US28’s constitutive activity. Though it remains disputable, and a likely answer to the Fractalkine-mediated decreased signalling may be ascribed to both a decrease in constitutive activity as well as an increase in ligand-receptor mediated internalization. Interestingly, CX3CL1 binding to US28 in fibroblasts also leads to activation of FAK via the same Gαq/11-protein involved in the ligand-independent activation of PLC-β but utilizes a different pathway. [1].

SMC (smooth muscle cells) migration however, is dependent on CC-chemokine binding to US28 and signalling via Gα12/13 to “FAK, ERK, RhoA and the actin cytoskeleton”[1]. Furthermore, CC-chemokine and Fractalkine binding both result in FAK activation, though through different G-proteins, Gα12 and Gαq respectively, and thus through different signalling pathways [13]. These two G-proteins have been shown to antagonize one another. For instance, Fractalkine signalling via Gαq is attenuated “in the presence of Gα12” and vice versa [13]. Lastly, the function of CC-chemokines and Fractalkine is cell type specific[13]. In SMC, Fractalkine inhibits CC-chemokine- induced migration. Conversely, Fractalkine induces migration in monocytes/macrophages, which RANTES (CC-chemokine) competitively inhibits in these cell types. Lastly, the βγ subunit also leads to stimulation of NF-κB thereby furthering viral dissemination. The pathophysiological relevance of Fractalkine and CC-chemokine binding and their cell type specificity will be further discussed in the Cardiovascular Disease section.

Taken together, the interaction of different ligands (CX3CL1 vs. CC-chemokines) to US28, or the absence thereof, within the same or among different cell types, can initiate distinct signalling pathways. Moreover, different activated forms of US28 exist and each form may interact with G-proteins at slightly different sites and thus contribute to “G-protein and ligand binding specificity”[1].

Regulation & Internalization/ recycling

Regulation of US28 constitutive activity

Generally, termination of signalling in agonist occupied GPCRs happens upon phosphorylation by GRK’s (G protein-coupled Receptor Kinases) and subsequent relocation of β-arrestins to the plasma membrane, thereby decoupling the G-proteins from their respective GPCRs. US28 is unique in that it undergoes constitutive phosphorylation and thus recruits β-arrestin independently of an agonist/ligand [14]. Mammalian GRK 2 and 5 carry out strong phosphorylation of Ser/thr residues in the carboxyl terminal tail of US28 of which serine 323 has been identified (through mutational studies) to be especially vital for signal attenuation and signalling [14]. The following binding of β-arrestin to the phosphorylated residues sterically inhibits the interaction between US28 and G-protein, resulting in signal termination seen experimentally as decreased signalling through Gαq-stimulated inositol phosphate pathway [14]. Besides phosphorylation, GRK2 contains an RGS domain (Regulator of G-Protein Signalling) that has affinity for Gαq subunit, which upon binding, blocks the interaction and consequently the activation of G-proteins thereby attenuating and terminating signalling [14]. GRK5 does not share same feature.

It may seem counter intuitive for a virus to stimulate constitutive phosphorylation and signal cessation. This mechanism however, may be a mean to prevent chronic signalling and maintain moderate level of signalling through which, the HCMV infected cell is able to evade recognition by the cellular apoptotic and defence apparatus [14].

Internalization and recycling of US28

US28 internalization happens primarily through a Clathrin-mediated endocytic pathway and to a lesser degree through a lipid raft & Caveolae mediated pathway [6]. The Clathrin-mediated pathway has been shown to be both β-arrestin dependent and β-arrestin independent (AP-2).

AP-2 (Adaptin) is an adaptor protein that binds directly to chemokine receptors through di-leucine motifs in the C-terminal domain, which subsequently recruits clathrin, forming clathrin-coated pits that “pinch off” to intracellular, clathrin coated vesicles via activated dynamin [7]. In US28, dileucine motifs at aa position 305/306 in the c-terminal, have been shown to affect early endocytosis [6]. The clathrin coated vesicles either enter the late endosomal compartment and fuse with lysosomes for degradation and/or enter the perinuclear-recycling compartment and are recy
cled back to the plasma membrane [7]. Although some articles ([6]), deny the importance of β-arrestin in the internalization process, much evidence points towards β-arrestin possessing a significant role in US28 internalization [15]. As mentioned above, β-arrestin is recruited following GRK phosphorylation of US28. Apart from affinity to phosphorylated residues, β-arrestin also has affinity for the β2-adaptin subunit of AP-2 and to clathrin. The association to clathrin and the consequent activation of dynamin results in the same internalization process as AP-2. Lastly, palmitoylation at the C-terminal of US28 has shown to “enhance GRK and β-arrestin recruitment” and therefore enhance US28 internalization [6]. Palmitoylation also exhibits great importance in the association of US28 to lipids rafts and caveolae [7].

Lipid raft and caveolae mediated endocytic pathway constitutes the alternative, clathrin-independent pathway in which US28 receptor is internalized. Lipid rafts are microdomains in the plasma membrane composed of glyco-sphingolipids and because of their resistance to solubilisation to detergents (NP-40 and Triton X), they are also known as “detergent resistant microdomains” [7]. Caveolae is a subfamily of lipid rafts and contain special marker proteins called caveolae 1-3 in addition to cholesterol and glyco-sphingolipids [7]. They may constitute a subdomain of the lipid rafts [6]. US28 has been localized within these detergent-resistant microdomains and are therefore internalized upon endocytosis of these domains. The endocytosis of US28 with lipid rafts and caveolae are facilitated by dynamin [6]. The resulting vesicles then undergo same fate as the clathrin coated vesicles by entering the early endosomal compartment and either fusing with lysosomes or entering the perinuclear-recycling compartment [7].

Taken together, HCMV infected cells undergo constitutive internalization and recycling as well as utilizing different endocytic pathways and may therefore serve as a decoy “for inflammatory chemokines”[6]. By constitutively internalizing receptors that have bound inflammatory chemokines (chemokine scavenging), the ability to activate and attract immune effector cells is impaired and thus enhances the vitality and dissemination of the HCMV.

Pathophysiological relevance of US28

Cardiovascular disease

The functional phenotypes of US28-expressing cells upon binding to CC-chemokines and Fractalkine in SMC and macrophages/monocytes respectively, has shown to accelerate and exacerbate cardiovascular disease [13]. As mentioned previously, RANTES and MCP-1 promotes SMC migration whereas Fractalkine inhibits it. The fractalkine-induced inhibition in SMC happens at a genetic level by reversing the “transcriptional activation required for cellular migration.” [13]. In practice, RANTES induces migration of HCMV and US28-expressing SMC from tunica media to inflammatory sites in tunica intima followed by Fractalkine-induced SMC-adhesion and accumulation in tunica intima [13]. Conversely, Fractalkine binding causes “robust migration” of US28-expressing macrophages and monocytes into atherosclerotic plaques leading to foam cell formation and thus contribute to vessel stenosis [13]. Additionally, locally produced cytokines such as IFN-γ, TNF-α and IL-1 recruit more inflammatory cells and increase the expression of membrane-bound Fractalkine thereby further aggravating cardiovascular disease [13]. All in all, US28 exploits two mechanisms in accelerating cardiovascular disease; CC-chemokine mediated SMC migration into intima and Fractalkine-mediated monocyte/macrophage migration into atherosclerotic lesions.

Oncogenesis

Viruses such as Human papilloma virus (HPV), Kaposi’s sarcoma-associated herpes virus (KSHV) and Epstein-Barr-virus (EBV) are all known to induce oncogenesis resulting in cervical cancer, Kaposi’s sarcoma and Burkitt’s lymphoma respectively [3]. Like so, there has been established a clear link between HCMV and tumorigenesis. HCMV infected cells are not able to cause and transform normal cells, but rather promote tumorigenesis in already neoplastic cells [3]. Evidence points toward the ability of US28 receptors’ constitutive activity (Gq) to stimulate cell cycle progression and angiogenesis as being one of the key causal factors in promoting tumorigenesis [3]. Apart from the constitutive activity of US28, the binding of ligands (ex. RANTES and MCP-1) and the resulting migratory capabilities can enhance the invasiveness of tumorigenic cells and thereby aggravate an already existing cancer [3].

The angiogenic phenotype is seen in vitro by increased secretion of VEGF in US28-WT expressing cells . VEGF promoter activation has shown to be accelerated through Gαq, Gβγ, P38 and P44/42 MAPK that stimulate downstream transcriptions factors . Maussang, D., et al (2006) carried out an experiment to measure VEGF plasma levels in three groups of (nude, NIH-3T3 transfected) mice: US28-WT, US28-R129A [G-protein uncoupled], and mock. US28-WT attained the highest plasma concentration, US28-R129A came second, and the mock measured the lowest, indicating that US28-WT constitutive activity results in increased VEGF plasma levels as well as the fact that angiogenesis also is [to a lesser degree] Gq-independent [3]. Furthermore, new blood vessel formation was identified in both US28-WT and US28-R129A using immunostaining against CD31[3].

Another characteristic of US28 constitutive activity is enhanced cell cycle progression. US28 expressing cells have shown to be more represented in the S and G2 phases and to a lesser extent the G1 phase [3]. Furthermore, up-regulation of Cyclin D1, responsible for G1 – S phase transition, is seen in US28-expressing cells. In another experiment of Maussang, D., et al (2006), nude mice that had been injected with NIH-3T3, US28-WT expressing cells all showed tumor formation at all inoculations sites after 3 weeks. In comparison, the US28-R129A [G-protein uncoupled] mice showed tumor formation after 6 weeks, and the mock failed to show tumor formation even after 75 days. RT-PCR was used to confirm US28 gene expression in all of the tumor cells. Thus US28 receptors’ constitutive activity is a prerequisite for both the increased cell cycle progression and angiogenesis. More specifically, COX-2 activation through US28 constitutive activity has been shown to be largely responsible [16].

COX-2 is an important mediator of inflammatory diseases and constitutes an important determinant in many cancer forms. It is often expressed in “premalignant lesions and malignant tumors” and is known to induce neoplasia [16]. Moreover, COX-2 is heavily expressed in US28-expressing cells and is enhanced at both mRNA and protein levels by NF-κB, which itself (as described earlier) is stimulated by both Gαq/11 and Gβγq/11 through US28 receptors’ constitutive activation of Gq/11 [16]. COX-2 induces the synthesis of PGEF-2 that auto-activates its cognate receptor (EP1-4) and leads to increased cell proliferation/tumor formation and angiogenesis by enhancing Cyclin D1 and VEGF transcription respectively. COX-2 activation is solely Gq/11 dependent [16]. Taken together, COX-2 is an important mediator in US28-mediated tumorigenesis.

Increased cell proliferation has also been ascribed to the expression of IL-6 induced by βγ subunit stimulation of NF-κB upon US28 receptor activation [17]. IL-6 binds to its cognate receptor (autocrine and paracrine fashion), thereupon activating a JAK1- STAT3 signalling axis, leading to the transcription of Cyclin D, COX, and VEGF, all of which accelerates cell proliferation. [17]

US28 is therefore an important determinant in tumorigenesis. In practice, colorectal cancer, the third most frequent and lethal cancer in the US, is coupled with an overexpression of US28, showing the aforementioned characteri
stics of enhanced cell cycle progression, angiogenesis and increased invasion potential [9]. Additionally, in immunocompromised cancer patients, a reactivation of HCMV may further aggravate the oncogenic potential of the virus through US28 [16]. Interestingly, US28-receptor has also shown to act as a co-receptor for HIV-1 entry, thus potentially causing an immunocompromised state [5]. In light of all this, US28 becomes an important, potential pharmacological target in lessening the pathological consequences of HCMV infected individuals.

US28 as pharmacological target

The pathological role of US28 in viral dissemination and thus secondarily exacerbating vascular disease, oncogenesis and HIV is both ligand dependent and ligand independent. Attenuating the respective G-protein-mediated signalling pathway by targeting US28 thus becomes a mean to which, the aforementioned pathological states can be ameliorated.

Small Molecules

The first and most studied small molecule inverse agonist on US28 constitutive signalling is VUF2774 [5]. This small non-peptidergic molecule was discovered through screening and subsequent testing of GPCR ligands for their capacity to modulate basal, constitutive activation of PLC-β [4, 5]. VUF2774 exhibits dose-dependent inhibition behaviour upon interacting with US28 in COS-7 cells [see figure 1] [5]. A 90% inhibition of the constitutive US28- PLC-β mediated InsP accumulation and an IC of 3.5µM were seen in these COS-7 cells (not ascribed to cellular toxicity) [5]. Additionally, in 293 T-cells, VUF2774 was able to inhibit HIV-1 entry into CD4+ cells by 60% at a concentration of 1µM [see figure 2 A&B] [5].

Contrary to chemokines, which are relatively large peptides and thus primarily binds to the N-terminus and extracellular loops of GPCRs, small molecules often bind to hydrophilic pockets within the transmembrane domains of GPCRs [1]. In fact, VUF2774 binds to the highly conserved glutamic acid residue Glu277 in the 7th TM of US28, possibly through a salt bridge to its positively charged piperidine moiety [5, 18]. In saturation binding assays, VUF2774 noncompetitively displaces 125I-RANTES binding to US28 in a dose-dependent manner [see figure 3A&B] [5]. Bmax decreased with increasing amounts of VUF2774 whilst Kd remained relatively the same. Taken together VUF2774 acts as an allosteric, noncompetitive inverse agonist.

The piperidine ring, the hydroxyl-group at the fourth position of the piperidine ring and the chlorosubstituent are important moieties in the activity of VUF2774, whereas a higher flexibility is accepted “at the benzhydryl moiety” (increased lipophilicity and removal of cyano-group) [see Table 1] [4, 5]. Hulshof, J.W. et al (2006) attempted to refine the VUF2774 lead compound structure by modifying the diphenyl group, propyl linker, and fourth position of the piperidine ring. As expected, modification of the diphenyl group yielded no effect in modulating US28 basal signalling. However, introducing a double bond between the piperidine moiety and the diphenyl group led to a 3-fold increase in binding affinity (IC50: 1.7 vs. 4.9 µM) [see table 2] [4]. Additionally, substituting the 4-hydroxy-group in the piperidine ring with a methylamine group yielded a 6-fold increase in affinity (IC50: 0.8 vs. 4.9 µM) [see table 3] [4]. Taken together, VUF2774 has become a promising scaffold in developing and discovering even more refined small molecule inverse agonists against US28. VUF2774 and its binding conformation with US28 has also been used to virtual screen and test commercially available small molecules compounds, such as the discovery of ZINC3854746, which exhibits a “twofold improved potency compared to VUF2774” [18].

Apart from VUF2774, other small molecules have been discovered over the years, including: Methiotepin (piperazinyldibenzothiepine derivative) and the structurally related oxoclothepin (antagonists that block CCL5 binding) [19], Di- and tetra-hydroisoquinolines (inverse agonists on constitutive PLC-β activation) [2], Biphenyl-derived amides (inverse agonists) [20], and flavonoid-based inverse agonists (on constitutive PLC-β activation) [21]. Yet other novel small molecules are emerging as potential modulators of US28 constitutive signalling.

FTP (Fusion Toxicity protein)

Immunotoxin protein, initially used as an anti-cancer therapy, is a new group of therapeutics that has big potential in killing HCMV infected cells by using US28 receptor as a mean of toxin delivery [22]. These protein-based therapeutics are chimeric molecules consisting of a toxin, often a fragment of the Pseudomonas aeruginosa exotoxin A (PE), fused to a target moiety that facilitates internalization of the toxin [23]. PE consists of three domains: domain I (Receptor-binding domain), domain II (translocation domain), domain Ib (unknown function), and domain III (enzymatic cytotoxic domain) [22]. It is the receptor-binding domain that can be replaced with another target domain and thus affect target selectivity. Once the fusion toxin protein binds to the target cell and is internalized, a subsequent cleavage of domain II leads to toxin release that eventually results in protein synthesis arrest and cellular apoptosis [figure 4][22, 23].

The high, constitutive, ligand-independent activity and internalization of US28 enables an extremely effective delivery of PE-toxins into US28-expressing cells and thus makes it a prime target for FTPs in killing HCMV-infected cells. CX3CL1 was used as targeting moiety in the first FTP created against US28-expressing cells due to its high affinity to US28 receptor (compared to CC-chemokines) even after replacement of the c-terminally attached mucin-like stalk with other proteins [22]. Furthermore CX3CL1-FTP exhibits high selectivity in that it only has affinity to US28 and the (endogenous) CX3CR1 receptor and thus is associated with potentially few off-target effects [22]. Interestingly, CX3CL1-FTP possesses a “80-fold higher affinity for US28 than for CX3CR1” [22]. In correlation to this, at 0.1 nM CX3CL1-FTP, 60% of US28-expressing cells were killed compared with only 10% of CX3CR1-expressing cells killed [22].

In an attempt to increase selectivity towards US28-expressing cells, Spiess, K., et al (2015) created 35 variants of CX3CL1-FTP with mutations in the CX3CL1-domain. Out of the 35 variants, F49A-FTP, with a single point mutation (Phe39 to Ala) in the CX3CL1 domain, showed the highest selectivity with an 182-fold increase in affinity to US28-receptor compared to CX3CR1 [figure 5]. Compared to Ganciclovir (GCV), the current “gold-standard drug” against HCMV, F49A-FTP resulted in a 2 x 104-fold higher potency in single-dose experiments (in vitro) [figure 6B]. Upon repeated treatment regiment with F49A-FTP, 1nM of F49A-FTP showed to be superior to 0.1mM GCV treatment [figure 6C]. Lastly, using bioluminescence signal from luciferase activity as indication of HCMV-replication in ToleduLUC-infected SCID-Hu mouse, F49A-FTP showed only “transient, weak signal on days 4-6 post-infection, which then dropped to background levels” and once again proved to be superior to GCV-treatment [22].

Spiess, K., et al (2017) attempted to refine F49A-FTP by synthesizing F49A-FTP analogues with modifcations in the chemokine domain, the linker region and the PE region. Although some changes in the chemokine domain led to higher affinity to US28 (F49L-FTP), an increased killing of CX3CR1-expressing cells were seen. Elongation and deletion of the linker region yielded a reduction in killing efficacy. Although changes in PE region improved cytotoxicity, it did so in both US28- and CX3CR1-expressing cells. Taken together, F49A-FTP remains to be the best candidate in treating HCMV in the context of selectivity and cytotoxicity combined (513-fold higher potency on US28- vs. CX3CR1-expressing cells) [23].

In the context of allo-haemotopoietic stem cell transplants (HSCT), F49A-FTP has shown to be highly effective in killing latent, HCMV-infected, early myeloid lineage cells [24]. This is of great importance, since reactivation HCMV in these cells is seen when they [early myoloid lineage cells] terminally differentiate into macrophages and DCs, potentially resulting in life-threatening states. Krishna, B.A., et al (2017) looked at the effect of F49A-FTP on experimental and natural, latently-infected, US28-expressing CD14+ monocytes and CD34+ progenitor cells. As “a proof of principle” and as expected, F49A-FTP led to a strong reduction of the aforementioned cells [24]. Furthermore, due to the nature of viral latency, F49A-FTP resistence is unlikely to occur in these types of cells. Although the affinity of F49A-FTP to CX3CR1 is very low, it yet remains. For this reason, F49A-FTP should not be given directly to recipients in HSCT but rather used to clear grafts (stemcells and solid organs) of latently infected cells before engraftment [24]. Taken together, F49A-FTP along with further refinements may become a new, highly effective therapeutic in transplantations as well as in the treatment of HCMV and HCMV-related diseases.

Conclusion

This review has covered the general two-site binding model for US28 using Fractalkine as an example and the functional selectivity in signalling pathways, which is dependent on the presence of ligand, the type of ligand, the absence of ligand on US28 altogether as well as cell type. Additionally, regulation and internalization has been discussed as being important factors for HCMV dissemination and chemokine scavenging. The functional, pathophysiological consequences of US28 and its associated signalling pathways have been examined and discussed in relevance to atherosclerosis in cardiovascular diseases, cell proliferation in tumorigenesis, and shortly, US28’s role in HIV entry of CD4+-co-expressing cells. Most importantly, US28 as a pharmacological target in preventing HCMV mediated diseases has been discussed. Variants of the small molecule, VUF2774, along with the FTP-variant, F49A, have shown to be the most promising candidates (in terms of affinity, efficacy and potency) in the development of novel pharmaceuticals against HCMV associated diseases. Further testing in animal-models and future testing in human trials remains to be carried out to gather a deeper insight into the yet unknown side effects of the drugs.

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