In recent years, entry inhibitors have emerged as a new class of antiviral drugs, with the HIV fusion-blocking peptide enfuvirtide being the first available fusion inhibitor applied in clinical treatment of a viral infection in humans (57
). Several small-molecule entry inhibitors against other important viruses, such as measles virus (35
), influenza virus (43
), respiratory syncytial virus (12
), and arenaviruses (22
), are currently in development. EBOV-GP mediates binding of EBOV with its cell surface receptor(s)/coreceptor(s) and subsequent entry, involving endocytosis of virus and fusion between viral and host cell membranes (44
). Therefore, we hypothesized that blocking of EBOV-GP-mediated viral entry will lead to inhibition of infection. Moreover, the aggressive nature of EBOV infections, in particular the rapid and overwhelming viral burdens in infected patients and EBOV-GP-related cell cytotoxicity (44
), justify preferentially targeting the entry process over subsequent downstream stages of viral replication.
In this study, we used HIV/EBOV-GP pseudotype virus as a surrogate model to screen libraries of 52,500 small-molecule compounds for inhibitors based on the following rationale. Since viral entry is determined solely by interaction of the virus envelope proteins with cell receptors (32
), the replication-incompetent HIV/EBOV-GP mimics the EBOV entry process (29
) and can be used in HTS of compound libraries in a BSL-2 laboratory. The HTS “hits” include at least four possible categories of inhibitors: (i) inhibitors of HIV/EBOV-GP entry, (ii) inhibitors of HIV replication, (iii) inhibitors of luciferase enzyme activity, and (iv) cytotoxic compounds. Therefore, to eliminate off-target hits such as the inhibitors of HIV replication and/or luciferase enzyme activity, we counterscreened the HTS hits with HIV/VSV-G pseudotype virus. HIV/VSV-G has the same HIV backbone as HIV/EBOV-GP but expresses VSV-G, a member of a different class of viral fusion-active protein (24
), on its surface. The counterscreen with HIV/VSV-G proved to be particularly useful since it eliminated the off-target hits that compromise 95% of the total HTS hits (Table ). It is possible that the counterscreen may have eliminated some HIV/EBOV-GP entry inhibitors, since the cell receptors of both EBOV-GP and VSV-G are not known and the wild-type EBOV and VSV infect a wide range of cells. Nevertheless, the counterscreen is an important step to eliminate undesired off-target HTS hits, and it allows us to focus on the most specific hits. Moreover, both EBOV and VSV enter cells by endocytosis followed by pH-dependent membrane fusion in endosomes (11
). Therefore, this counterscreen also eliminated compounds that modulate membrane trafficking or endosomal pH and identified compounds that bind to either EBOV-GP or specific components of the EBOV entry pathway that are not shared with VSV.
We identified eight novel inhibitors of EBOV entry. The IC90 values for six of the eight compounds against GFP-ZEBOV were within ~2- to 3-fold of the values for HIV/EBOV-GP, while IC90 values for compounds 3 and 4 against GFP-ZEBOV are significantly higher (>20-fold difference) (Table ). At this time we do not know the reasons for the differences in the IC90 values between HIV/EBOV-GP and GFP-ZEBOV, but they may be due to differences in the (i) virus morphology (EBOV is cylindrical, while HIV/EBOV-GP is spherical), (ii) GP density at the cell surface, (iii) GP modification (e.g., producer cell type-specific glycosylation patterns), and (iv) target cells (293T versus VeroE6). Compound 7 was selected for further chemical optimization and mechanism studies because (i) the benzodiazepine structure is a suitable “drug-like” starting point for medicinal chemistry, (ii) the compound exhibited good potency in both the HIV/EBOV-GP and cell culture grown-infectious EBOV assay, and (iii) its selectivity index (SI of >5) indicates that the compound exhibits significant selectivity for antiviral activity versus cytotoxicity (Fig. ). The preliminary SAR evaluation so far has identified several additional benzodiazepine analogs that exhibit anti-EBOV activity, including one more potent (3 times more) analog (compound 12) that also displays a selectivity index of greater than 10. Although the sample size for the SAR analysis is small, the increased activity of compound 12 suggests that more potent inhibitors can be designed using the benzodiazepine backbone. In addition, with a selectivity index of >10 and higher antiviral potency, compound 12 will be the starting point for optimization of the benzodiazepine series to identify a lead inhibitor using medicinal chemistry. The systematic synthesis of a larger library of compounds (see the supplemental material), with the substituents carefully controlled, will be necessary to identify informative SAR trends, and this will be the first priority of the synthetic plan for further development of the benzodiazepine series to obtain lead compounds.
Virus entry is a multistep process, and target identification for compound 7 may be challenging. Three approaches have been taken so far to determine the antiviral target. First, we examined the antiviral spectrum of compound 7 against various filoviruses and nonfiloviruses (Table ). It is reasonable to hypothesize that a compound capable of inhibiting multiple viruses from different families most likely inhibits or blocks either host receptors or other host factors involved in virus entry. However, compound 7 inhibited primarily only EBOV and MARV, the two members of the filovirus family. The critical amino acid residues that are important for virus entry are conserved in both EBOV-GP and MARV-GP (29
). Compound 7 exhibited very weak activity against other viruses bearing similar type 1 envelope proteins but, in general, exhibited no activity against other RNA and DNA viruses. The results suggest that compound 7 is not inhibiting host factors such as the endosomal pathways involved in infection of a number of enveloped viruses.
Second, we performed “time-of-addition” studies, which indicated that compound 7 blocks at an early stage of virus entry into cells, possibly by binding to EBOV-GP. Preincubation of HIV/EBOV-GP with compound 7 also blocked its infection of 293T cells (data not shown), further suggesting that compound 7 binds to HIV/EBOV-GP. Similarly, the differential binding of HIV/EBOV-GP and HIV/VSV-G by compound 7 in the virus-compound binding assay suggests that compound 7 binds EBOV-GP, since both the viruses have the same HIV core. Taken together, these studies indicate that compound 7 acts early in the infection process, probably by binding to EBOV-GP. From this experiment, it is not possible to stoichiometrically measure the binding of EBOV-GP and compound 7. Since the pseudotype virus randomly acquires EBOV-GP from the surfaces of infected cells and since it is competent for only one round of replication, it has not been feasible to determine precisely the number of mature trimeric EBOV-GP molecules on the viral surface, the number of viral particles carrying the EBOV-GP, or the stoichiometry of the compound 7-EBOV-GP interaction. Nuclear magnetic resonance (NMR) studies are planned to better understand the interactions of compound 7 with EBOV-GP.
EBOV-GP is heavily glycosylated by both N-linked and O-linked glycans. As a result, only a few sites are left exposed and accessible for binding interactions. These sites include (i) a region at the base of the chalice where GP1 meets GP2, (ii) short linear stretches of polypeptide between glycans in the mucin-like domain, and (iii) the HR2 region. Studies using an EBOV-GP1 RBD peptide (amino acids [aa] 54 to 201) showed that compound 7 has no effect on the binding of EBOV-GP to 293T and Vero cell lines. In addition, compound 7 inhibited both wt HIV/EBOV-GP and HIV/EBOV-GPΔmucin with similar potencies (data not shown). In HIV/EBOV-GPΔmucin, the mucin domain is removed to expose the EBOV-GP1 RBD. Taken together, these results indicate that the primary mechanism of action of compound 7 is not direct blocking of virus attachment to the cells by binding the RBD of GP1. Instead, we hypothesize that compound 7 binds to a region of the EBOV-GP at the base of the chalice where GP1 meets GP2, since this region is exposed in both EBOV-GP and EBOV-GPΔmucin.
Supporting evidence for this hypothesis comes from our computational studies using the recently published crystal structure (3.4 Å) of EBOV-GP in its metastable trimeric, prefusion conformation (25
), as confirmed by our mutational studies. The computational studies using the Schrödinger SiteMap program identified a putative hydrophobic binding pocket (S2) for small-molecule ligand binding at the junction of the GP1 base subdomain with the GP2 internal fusion loop and the heptad repeat 1A helix (HR1A
). Amino acid residues Val66, Leu68, Asn69, Leu70, Leu184, and Leu186 from the base and head subdomains of GP1 together with residues Tyr517, Met548, Leu554, and Leu558 on the GP2 internal fusion loop in the hydrophobic S2 binding site appear to be involved in interacting with bound ligands, according to the model. Indeed, a significant reduction in the potency of compound 7 against HIV/EBOV-GP mutants N69A, L70A, L184A, I185A, L186A, and K190A/K191A further implicates the S2 hydrophobic pocket of EBOV-GP as a potential binding site for small-molecule ligands such as the benzodiazepine compound 7.
The EBOV-GP1 base subdomain contains four discontinuous sections (residues 33 to 69, 95 to 104, 158 to 167, and 176 to 189) which form two mixed beta-sheets, with strands β3 and β13 shared between the two beta-sheets. The head subdomain is composed of residues from four discontinuous segments, i.e., residues 70 to 94, 105 to 157, 168 to 175, and 214 to 226, and forms a four-stranded, mixed beta-sheet supported by an alpha-helix and a smaller, two-stranded antiparallel beta-sheet (25
). As shown from the mutation studies, the HIV/EBOV-GP N69A and L70A mutants are not inhibited by compound 7, suggesting that the amino acid residues at the junction between the head and base subdomains of GP1 are important for binding. Compound 7 weakly inhibited HIV/LASV-GP, HIV/LMCV-GP, HIV/MACV-GP, and HIV/HA(H5) at higher concentrations. These pseudotype viruses also contain type 1 membrane proteins similar to EBOV-GP. The type 1 membrane protein carries a similar cavity formed by the clamping of the N-terminal membrane-proximal base of the receptor binding subunit over the internal fusion loop through hydrophobic interactions, including an interchain disulfide bond (37
). Therefore, our results suggest that the size and the conformation of the S2 hydrophobic pocket of EBOV-GP are important and that compound 7 selectively binds in this pocket to prevent viral activity. This hypothesis is supported by limited preliminary SAR analysis of compound 7, which showed that bulky aromatic substitutions on the diazepine ring introduce some size constraints on the inhibitor and reduce its antiviral activity (Fig. ; Table ). The lack of activity for compounds with a bulky substituent(s) implies a binding site with well-defined boundaries.
A limited number of small-molecule inhibitors of EBOV have been discovered (7
). However, none of these inhibitors are used in clinical settings, and none of them resemble compound 7 structurally. The existing small-molecule inhibitors of EBOV infections can be characterized by three general modes of action: (i) impairment of viral replication, (ii) stimulation of innate antiviral mechanisms, and (iii) prevention of virus entry into the cells.
The carbocyclic adenosine analog 3-deazaadenosine (C-c3Ado), inhibits cellular S
-adenosylhomocysteine hydrolase and inhibits the replication of EBOV Zaire in vitro
, with an IC50
of 30 μM (7
). The activity of this antiviral agent has been attributed to diminished methylation of the 5′ cap of viral mRNA by (guanine 7) methyltransferase, which impairs the translation of viral transcripts. Administration of C-c3Ado to EBOV-infected mice has also been found to dramatically increase production of alpha interferon (IFN-α), which may serve to counteract the virus suppression of the innate antiviral response. Unfortunately, C-c3Ado failed to promote IFN-α production in Ebola virus-infected monkeys (7
). More recently, other small-molecule EBOV inhibitors, such as FGI-103, FGI-104, and FGI-106, were discovered (1
) by HTS screening. These inhibitors were found to be potent and to provide protection against EBOV and Marburg virus in vitro
, and in vivo
. While FGI-103 inhibits infection by an unknown mechanism, FGI-104 targets EBOV vp40-tgs101-mediated budding. In contrast, FGI-106 displays potent and broad-spectrum inhibition of lethal viral hemorrhagic fever pathogens, including Ebola, Rift Valley and dengue fever viruses, in cell-based assays, suggesting that it interferes with a common pathway utilized by different viruses (1
The glycodendritic structure BH30sucMan (23
), which contains 32 individual α-mannose units linked to the hyperbranched dendrimer BH30 through succinyl spacers, has recently been shown to block the interaction between dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) (58
) and EBOV-GP. However, this compound is not specific for EBOV infection. Endosomal proteases CatB and CatL mediate viral entry by carrying out proteolysis of the EBOV-GP1 subunit (11
). Recently, the CatL inhibitor tetrahydroquinoline oxocarbazate was reported to inhibit EBOV infection at nanomolar concentrations (47
). It also inhibits other viruses that use CatL for entry. Unfortunately, given the demonstrated hypersensitivity of EBOV-GP1 to digestion by other proteases (17
), such as thermolysin (17
), the clinical prospects for antiviral agents that solely target CatB and CatL are not encouraging. Compound 7 and its analogs were found to have no activity against CatL in in vitro
enzymatic assays (data not shown). Compound 7 and its analogs differ from these previously reported small-molecule inhibitors by the specificity exhibited for filoviruses and the apparent mechanism of action. Unlike the other entry inhibitors, the benzodiazopenes may bind directly to EBOV-GP within a hydrophobic pocket at the EBOV GP1-GP2 interface. Moreover, blocking of propagation of EBOV at an early stage will minimize the chance for the virus to evolve and acquire drug resistance.
We conclude that compound 7 acts at an early stage of viral entry, apparently by binding to a hydrophobic pocket (S2) in the prefusion conformation of EBOV-GP and preventing infection by an unknown mechanism. An analogy can be made with several different classes of small-molecule HIV entry inhibitors, including maraviroc, that are thought to bind within a pocket created by four transmembrane domains of CCR5, an important HIV coreceptor (19
). Furthermore, computational and mutational studies suggest that the S2 hydrophobic binding pocket is a well-defined small-molecule binding site, which may serve as a viable target for future antifilovirus drug discovery.