Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

Molecular Characterization and Identification of Potential Inhibitors for 'E' Protein of Dengue Virus.

Dengue is an arthropod-borne acute febrile illness caused by Dengue Virus (DENV), a member of Flaviviridae. Severity of the infection ranges from mild self-limiting illness to severe life-threatening hemorrhagic fever (DHF) and dengue shock syndrome (DSS). To date, there is no specific antiviral therapy established to treat the infection. The current study reports the epidemiology of DENV infections and potential inhibitors of DENV 'E' protein. Among the various serotypes, DENV-2 serotype was observed more frequently, followed by DENV-4, DENV-1, and DENV-3. New variants of existing genotypes were observed in DENV-1, 2, and 4 serotypes. Predominantly, the severe form of dengue was attributable to DENV-2 infections, and the incidence was more common in males and pediatric populations. Both the incidence and the disease severity were more common among the residents of non-urban environments. Due to the predominantly self-limiting nature of primary dengue infection and folk medicine practices of non-urban populations, we observed a greater number of secondary dengue cases than primary dengue cases. Hemorrhagic manifestations were more in secondary dengue in particularly in the pediatric group. Through different computational methods, ligands RGBLD1, RGBLD2, RGBLD3, and RGBLD4 are proposed as potential inhibitors in silico against DENV-1, -2, -3, and -4 serotypes.

Two Distinct Lysosomal Targeting Strategies Afford Trojan Horse Antibodies With Pan-Filovirus Activity.

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability-the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a 'Trojan horse' therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses.

Development of peptides targeting receptor binding site of the envelope glycoprotein to contain the West Nile virus infection.

West Nile virus (WNV), re-emerging neurotropic flavivirus, can cross the blood-brain barrier (BBB) and cause fatal encephalitis and meningitis. Infection of the human brain microvascular endothelial cells (hBMECs), building blocks of the BBB, represents the pivotal step in neuroinvasion. Domain III (DIII) of the envelope (E) glycoprotein is a key receptor-binding domain, thus, it is an attractive target for anti-flavivirus strategies. Here, two combinatorial phage display peptide libraries, Ph.D.-C7C and Ph.D.-12, were panned against receptor-binding site (RBS) on DIII to isolate peptides that could block DIII. From series of pannings, nine peptides (seven 7-mer cyclic and two 12-mer linear) were selected and overexpressed in E. coli SHuffle T5. Presence of disulfide bond in 7-mer peptides was confirmed with thiol-reactive maleimide labeling. Except for linear peptide 19 (HYSWSWIAYSPG), all peptides proved to be DIII binders. Among all peptides, 4 cyclic peptides (CTKTDVHFC, CIHSSTRAC, CTYENHRTC, and CLAQSHPLC) showed significant blocking of the interaction between DIII and hBMECs, and ability to neutralize infection in cultured cells. None of these peptides showed toxic or hemolytic activity. Peptides identified in this study may serve as potential candidates for the development of novel antiviral therapeutics against WNV.

Attacking COVID-19 Progression Using Multi-Drug Therapy for Synergetic Target Engagement.

COVID-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. Over the past 12 months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has already infected over 160 million (>20% located in United States) and killed more than 3.3 million people around the world (>20% deaths in USA). As we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack SARS-CoV-2 on multiple fronts. We have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential SARS-CoV-2 viral proteins and their host protein interactors-S/Ace2, Tmprss2, Cathepsins L and K, and Mpro-to prevent binding, membrane fusion and replication of the virus, respectively. All together, we generated an ensemble of structural conformations that increase high-quality docking outcomes to screen over >6 million compounds including all FDA-approved drugs, drugs under clinical trial (>3000) and an additional >30 million selected chemotypes from fragment libraries. Our results yielded an initial set of 350 high-value compounds from both new and FDA-approved compounds that can now be tested experimentally in appropriate biological model systems. We anticipate that our results will initiate screening campaigns and accelerate the discovery of COVID-19 treatments.

Poly-l-lysine Glycoconjugates Inhibit DC-SIGN-mediated Attachment of Pandemic Viruses.

The C-type lectin receptor DC-SIGN mediates interactions with envelope glycoproteins of many viruses such as SARS-CoV-2, ebola, and HIV and contributes to virus internalization and dissemination. In the context of the recent SARS-CoV-2 pandemic, involvement of DC-SIGN has been linked to severe cases of COVID-19. Inhibition of the interaction between DC-SIGN and viral glycoproteins has the potential to generate broad spectrum antiviral agents. Here, we demonstrate that mannose-functionalized poly-l-lysine glycoconjugates efficiently inhibit the attachment of viral glycoproteins to DC-SIGN-presenting cells with picomolar affinity. Treatment of these cells leads to prolonged receptor internalization and inhibition of virus binding for up to 6 h. Furthermore, the polymers are fully bio-compatible and readily cleared by target cells. The thermodynamic analysis of the multivalent interactions reveals enhanced enthalpy-driven affinities and promising perspectives for the future development of multivalent therapeutics.

Adamantane derivatives as potential inhibitors of p37 major envelope protein and poxvirus reproduction. Design, synthesis and antiviral activity.

Currently, smallpox, caused by the variola virus belonging to the poxvirus family, has been completely eradicated according to the WHO. However, other representatives of poxviruses, such as vaccinia virus, cowpox virus, ectromelia virus, monkeypox virus, mousepox virus and others, remain in the natural environment and can infect both animals and humans. The pathogens of animal diseases, belonging to the category with a high epidemic risk, have already caused several outbreaks among humans, and can, in an unfavorable combination of circumstances, cause not only an epidemic, but also a pandemic. Despite the fact that there are protocols for the treatment of poxvirus infections, the targeted design of new drugs will increase their availability and expand the arsenal of antiviral chemotherapeutic agents. One of the potential targets of poxviruses is the p37 protein, which is a tecovirimat target. This protein is relatively small, has no homologs among proteins of humans and other mammals and is necessary for the replication of viral particles, which makes it attractive target for virtual screening. Using the I-TASSER modelling and molecular dynamics refinement the p37 orthopox virus protein model was obtained and its was confirmed by ramachandran plot analysis and superimposition of the model with the template protein with similar function. A virtual library of adamantane containing compounds was generated and a number of potential inhibitors were chosen from virtual library using molecular docking. Several compounds bearing adamantane moiety were synthesized and their biological activity was tested in vitro on vaccinia, cowpox and mousepox viruses. The new compounds inhibiting vaccinia virus replication with IC50 concentrations between 0.133 and 0.515 µM were found as a result of the research. The applied approach can be useful in the search of new inhibitors of orthopox reproduction. The proposed approach may be suitable for the design of new poxvirus inhibitors containing cage structural moiety.

CX3CR1 Is a Receptor for Human Respiratory Syncytial Virus in Cotton Rats.

Respiratory syncytial virus (RSV) has been reported to use CX3CR1 in vitro as a receptor on cultured primary human airway epithelial cultures. To evaluate CX3CR1 as the receptor for RSV in vivo, we used the cotton rat animal model because of its high permissiveness for RSV infection. Sequencing the cotton rat CX3CR1 gene revealed 91% amino acid similarity to human CX3CR1. Previous work found that RSV binds to CX3CR1 via its attachment glycoprotein (G protein) to infect primary human airway cultures. To determine whether CX3CR1-G protein interaction is necessary for RSV infection, recombinant RSVs containing mutations in the CX3CR1 binding site of the G protein were tested in cotton rats. In contrast to wild-type virus, viral mutants did not grow in the lungs of cotton rats. When RSV was incubated with an antibody blocking the CX3CR1 binding site of G protein and subsequently inoculated intranasally into cotton rats, no virus was found in the lungs 4 days postinfection. In contrast, growth of RSV was not affected after preincubation with heparan sulfate (the receptor for RSV on immortalized cell lines). A reduction in CX3CR1 expression in the cotton rat lung through the use of peptide-conjugated morpholino oligomers led to a 10-fold reduction in RSV titers at day 4 postinfection. In summary, these results indicate that CX3CR1 functions as a receptor for RSV in cotton rats and, in combination with data from human airway epithelial cell cultures, strongly suggest that CX3CR1 is a primary receptor for naturally acquired RSV infection. IMPORTANCE The knowledge about a virus receptor is useful to better understand the uptake of a virus into a cell and potentially develop antivirals directed against either the receptor molecule on the cell or the receptor-binding protein of the virus. Among a number of potential receptor proteins, human CX3CR1 has been demonstrated to act as a receptor for respiratory syncytial virus (RSV) on human epithelial cells in tissue culture. Here, we report that the cotton rat CX3CR1, which is similar to the human molecule, acts as a receptor in vivo. This study strengthens the argument that CX3CR1 is a receptor molecule for RSV.

Discovery of a novel highly potent broad-spectrum heterocyclic chemical series of arenavirus cell entry inhibitors.

We identified and explored the structure-activity relationship (SAR) of a novel heterocyclic chemical series of arenavirus cell entry inhibitors. Optimized lead compounds, including diphenyl-substituted imidazo[1,2-a]pyridines, benzimidazoles, and benzotriazoles exhibited low to sub-nanomolar potency against both pseudotyped and infectious Old and New World arenaviruses, attractive metabolic stability in human and most nonhuman liver microsomes as well as a lack of hERG K + channel or CYP enzyme inhibition. Moreover, the straightforward synthesis of several lead compounds (e.g., the simple high yield 3-step synthesis of imidazo[1,2-a]pyridine 37) could provide a cost-effective broad-spectrum arenavirus therapeutic that may help to minimize the cost-prohibitive burdens associated with treatments for emerging viruses in economically challenged geographical settings.

Discovery of a Potent and Selective Chikungunya Virus Envelope Protein Inhibitor through Computer-Aided Drug Design.

The worldwide expansion of chikungunya virus (CHIKV) into tropical and subtropical areas in the last 15 years has posed a currently unmet need for vaccines and therapeutics. The E2-E1 envelope glycoprotein complex binds receptors on the host cell and promotes membrane fusion during CHIKV entry, thus constituting an attractive target for the development of antiviral drugs. In order to identify CHIKV antivirals acting through inhibition of the envelope glycoprotein complex function, our first approach was to search for amenable druggable sites within the E2-E1 heterodimer. We identified a pocket located in the interface between E2 and E1 around the fusion loop. Then, via a structure-based virtual screening approach and in vitro assay of antiviral activity, we identified compound 7 as a specific inhibitor of CHIKV. Through a lead optimization process, we obtained compound 11 that demonstrated increased antiviral activity and low cytotoxicity (EC50 1.6 µM, CC50 56.0 µM). Molecular dynamics simulations were carried out and described a possible interaction pattern of compound 11 and the E1-E2 dimer that could be useful for further optimization. As expected from target site selection, compound 11 inhibited virus internalization during CHIKV entry. In addition, virus populations resistant to compound 11 included mutation E2-P173S, which mapped to the proposed binding pocket, and second site mutation E1-Y24H. Construction of recombinant viruses showed that these mutations conferred antiviral resistance in the parental background. Finally, compound 11 presents acceptable solubility values and is chemically and enzymatically stable in different media. Altogether, these findings uncover a suitable pocket for the design of CHIKV entry inhibitors with promising antiviral activity and pharmacological profiles.

Convergence of a common solution for broad ebolavirus neutralization by glycan cap-directed human antibodies.

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form ß-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies.

Antivirals Targeting the Surface Glycoproteins of Influenza Virus: Mechanisms of Action and Resistance.

Hemagglutinin and neuraminidase, which constitute the glycoprotein spikes expressed on the surface of influenza A and B viruses, are the most exposed parts of the virus and play critical roles in the viral lifecycle. As such, they make prominent targets for the immune response and antiviral drugs. Neuraminidase inhibitors, particularly oseltamivir, constitute the most commonly used antivirals against influenza viruses, and they have proved their clinical utility against seasonal and emerging influenza viruses. However, the emergence of resistant strains remains a constant threat and consideration. Antivirals targeting the hemagglutinin protein are relatively new and have yet to gain global use but are proving to be effective additions to the antiviral repertoire, with a relatively high threshold for the emergence of resistance. Here we review antiviral drugs, both approved for clinical use and under investigation, that target the influenza virus hemagglutinin and neuraminidase proteins, focusing on their mechanisms of action and the emergence of resistance to them.

Blockers of the SARS-CoV-2 3a Channel Identified by Targeted Drug Repurposing.

The etiological agent of the COVID-19 pandemic is SARS-CoV-2. As a member of the Coronaviridae, the enveloped pathogen has several membrane proteins, of which two, E and 3a, were suggested to function as ion channels. In an effort to increase our treatment options, alongside providing new research tools, we have sought to inhibit the 3a channel by targeted drug repurposing. To that end, using three bacteria-based assays, we screened a library of 2839 approved-for-human-use drugs and identified the following potential channel-blockers: Capreomycin, Pentamidine, Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine and Fludarabine. The stage is now set for examining the activity of these compounds in detailed electrophysiological studies and their impact on the whole virus with appropriate biosafety measures.

Antivirals with common targets against highly pathogenic viruses.

Historically, emerging viruses appear constantly and have cost millions of human lives. Currently, climate change and intense globalization have created favorable conditions for viral transmission. Therefore, effective antivirals, especially those targeting the conserved protein in multiple unrelated viruses, such as the compounds targeting RNA-dependent RNA polymerase, are urgently needed to combat more emerging and re-emerging viruses in the future. Here we reviewed the development of antivirals with common targets, including those against the same protein across viruses, or the same viral function, to provide clues for development of antivirals for future epidemics.

Identification of Potential HCV Inhibitors Based on the Interaction of Epigallocatechin-3-Gallate with Viral Envelope Proteins.

Hepatitis C is affecting millions of people around the globe annually, which leads to death in very high numbers. After many years of research, hepatitis C virus (HCV) remains a serious threat to the human population and needs proper management. The in silico approach in the drug discovery process is an efficient method in identifying inhibitors for various diseases. In our study, the interaction between Epigallocatechin-3-gallate, a component of green tea, and envelope glycoprotein E2 of HCV is evaluated. Epigallocatechin-3-gallate is the most promising polyphenol approved through cell culture analysis that can inhibit the entry of HCV. Therefore, various in silico techniques have been employed to find out other potential inhibitors that can behave as EGCG. Thus, the homology modelling of E2 protein was performed. The potential lead molecules were predicted using ligand-based as well as structure-based virtual screening methods. The compounds obtained were then screened through PyRx. The drugs obtained were ranked based on their binding affinities. Furthermore, the docking of the topmost drugs was performed by AutoDock Vina, while its 2D interactions were plotted in LigPlot+. The lead compound mms02387687 (2-[[5-[(4-ethylphenoxy) methyl]-4-prop-2-enyl-1,2,4-triazol-3-yl] sulfanyl]-N-[3(trifluoromethyl) phenyl] acetamide) was ranked on top, and we believe it can serve as a drug against HCV in the future, owing to experimental validation.

Thiophen urea derivatives as a new class of hepatitis C virus entry inhibitors.

To develop unique small-molecule inhibitors of hepatitis C virus (HCV), thiophen urea (TU) derivatives were synthesised and screened for HCV entry inhibitory activities. Among them, seven TU compounds exhibited portent anti-viral activities against genotypes 1/2 (EC50 < 30 nM) and subsequently, they were further investigated; based on the pharmacological, metabolic, pharmacokinetic, and safety profiles, J2H-1701 was selected as the optimised lead compound as an HCV entry inhibitor. J2H-1701 possesses effective multi-genotypic antiviral activity. The docking results suggested the potential interaction of J2H-1701 with the HCV E2 glycoprotein. These results suggest that J2H-1701 can be a potential candidate drug for the development of HCV entry inhibitors.

In silico identification of Tretinoin as a SARS-CoV-2 envelope (E) protein ion channel inhibitor.

Viroporins are oligomeric, pore forming, viral proteins that play critical roles in the life cycle of pathogenic viruses. Viroporins like HIV-1 Vpu, Alphavirus 6 K, Influenza M2, HCV p7, and Picornavirus 2B, form discrete aqueous passageways which mediate ion and small molecule transport in infected cells. The alterations in host membrane structures induced by viroporins is essential for key steps in the virus life cycle like entry, replication and egress. Any disruption in viroporin functionality severely compromises viral pathogenesis. The envelope (E) protein encoded by coronaviruses is a viroporin with ion channel activity and has been shown to be crucial for the assembly and pathophysiology of coronaviruses. We used a combination of virtual database screening, molecular docking, all-atom molecular dynamics simulation and MM-PBSA analysis to test four FDA approved drugs - Tretinoin, Mefenamic Acid, Ondansetron and Artemether - as potential inhibitors of ion channels formed by SARS-CoV-2 E protein. Interaction and binding energy analysis showed that electrostatic interactions and polar solvation energy were the major driving forces for binding of the drugs, with Tretinoin being the most promising inhibitor. Tretinoin bound within the lumen of the channel formed by E protein, which is lined by hydrophobic residues like Phe, Val and Ala, indicating its potential for blocking the channel and inhibiting the viroporin functionality of E. In control simulations, tretinoin demonstrated a lower binding energy with a known target as compared to SARS-CoV-2 E protein. This work thus highlights the possibility of exploring Tretinoin as a potential SARS-CoV-2 E protein ion channel blocker and virus assembly inhibitor, which could be an important therapeutic strategy in the treatment for coronaviruses.

MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, and Avian Influenza Virus H5N1 Hemagglutinin Maturation.

Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions.IMPORTANCE Enveloped viruses express three classes of fusion proteins that are required for their entry into host cells via mediating virus and cell membrane fusion. Class I fusion proteins are produced from influenza viruses, retroviruses, Ebola viruses, and coronaviruses. They are first synthesized as a type I transmembrane polypeptide precursor that is subsequently glycosylated and oligomerized. Most of these precursors are cleaved en route to the plasma membrane by a cellular protease furin in the late secretory pathway, generating the trimeric N-terminal receptor-binding and C-terminal fusion subunits. Here, we show that a cellular protein, MARCH8, specifically inhibits the furin-mediated cleavage of EBOV GP, HIV-1 Env, and H5N1 HA. Further analyses uncovered that MARCH8 blocked the EBOV GP glycosylation in the Golgi and inhibited its transport from the Golgi to the plasma membrane. Thus, MARCH8 has a very broad antiviral activity by specifically inactivating different viral fusion proteins.

Harringtonine Inhibits Zika Virus Infection through Multiple Mechanisms.

Mosquito-borne Zika virus (ZIKV) is a Flavivirus that came under intense study from 2014 to 2016 for its well-known ability to cause congenital microcephaly in fetuses and neurological Guillain-Barré disease in adults. Substantial research on screening antiviral agents against ZIKV and preventing ZIKV infection are globally underway, but Food and Drug Administration (FDA)-approved treatments are not available yet. Compounds from Chinese medicinal herbs may offer an opportunity for potential therapies for anti-ZIKV infection. In this study, we evaluated the antiviral efficacy of harringtonine against ZIKV. Harringtonine possessed anti-ZIKV properties against the binding, entry, replication, and release stage through the virus life cycle. In addition, harringtonine have strong virucidal effects in ZIKV and exhibited prophylaxis antiviral ability prior ZIKV infection. The antiviral activity also observed in the treatment against Japanese encephalitis reporter virus (RP9-GFP strain). Overall, this study demonstrated that harringtonine would be a favorable potential candidate for the development of anti-ZIKV infection therapies.

Identification of Zika Virus Inhibitors Using Homology Modeling and Similarity-Based Screening to Target Glycoprotein E.

The World Health Organization has designated Zika virus (ZIKV) as a dangerous, mosquito-borne pathogen that can cause severe developmental defects. The primary goal of this work was identification of small molecules as potential ZIKV inhibitors that target the viral envelope glycoprotein (ZIKV E) involved in membrane fusion and viral entry. A homology model of ZIKV E containing the small molecule ß-octyl glucoside (BOG) was constructed, on the basis of an analogous X-ray structure from dengue virus, and >4 million commercially available compounds were computationally screened using the program DOCK6. A key feature of the screen involved the use of similarity-based scoring to identify inhibitor candidates that make similar interaction energy patterns (molecular footprints) as the BOG reference. Fifty-three prioritized compounds underwent experimental testing using cytotoxicity, cell viability, and tissue culture infectious dose 50% (TCID50) assays. Encouragingly, relative to a known control (NITD008), six compounds were active in both the cell viability assay and the TCID50 infectivity assay, and they showed activity in a third caspase activity assay. In particular, compounds 8 and 15 (tested at 25 µM) and compound 43 (tested at 10 µM) appeared to provide significant protection to infected cells, indicative of anti-ZIKV activity. Overall, the study highlights how similarity-based scoring can be leveraged to computationally identify potential ZIKV E inhibitors that mimic a known reference (in this case BOG), and the experimentally verified hits provide a strong starting point for further refinement and optimization efforts.