Inhibition Of Membrane Fusion Research Articles

Translation of Mycobacterium Survival Strategy to Develop a Lipo-peptide based Fusion Inhibitor*.

The entry of enveloped virus requires the fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such membrane fusion may emerge as a broad‐spectrum antiviral agent against a wide range of viral infections. Mycobacterium survives inside the phagosome by inhibiting phagosome–lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40‐repeat protein suggest that the trp‐asp (WD) sequence is placed at distorted β‐meander motif (more exposed) in coronin 1. The unique structural feature of coronin 1 was explored to identify a simple lipo‐peptide sequence (myr‐WD), which effectively inhibits membrane fusion by modulating the interfacial order, water penetration, and surface potential. The mycobacterium inspired lipo‐dipeptide was successfully tested to combat type 1 influenza virus (H1N1) and murine coronavirus infections as a potential broad‐spectrum antiviral agent.

Translation of Mycobacterium Survival Strategy to Develop a Lipo‐peptide based Fusion Inhibitor**

AbstractThe entry of enveloped virus requires the fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such membrane fusion may emerge as a broad‐spectrum antiviral agent against a wide range of viral infections. Mycobacterium survives inside the phagosome by inhibiting phagosome–lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40‐repeat protein suggest that the trp‐asp (WD) sequence is placed at distorted β‐meander motif (more exposed) in coronin 1. The unique structural feature of coronin 1 was explored to identify a simple lipo‐peptide sequence (myr‐WD), which effectively inhibits membrane fusion by modulating the interfacial order, water penetration, and surface potential. The mycobacterium inspired lipo‐dipeptide was successfully tested to combat type 1 influenza virus (H1N1) and murine coronavirus infections as a potential broad‐spectrum antiviral agent.

Antiviral Screen against Canine Distemper Virus-Induced Membrane Fusion Activity.

Canine distemper virus (CDV), a close relative of the human pathogen measles virus (MeV), is an enveloped, negative sense RNA virus that belongs to the genus Morbillivirus and causes severe diseases in dogs and other carnivores. Although the vaccination is available as a preventive measure against the disease, the occasional vaccination failure highlights the importance of therapeutic alternatives such as antivirals against CDV. The morbilliviral cell entry system relies on two interacting envelope glycoproteins: the attachment (H) and fusion (F) proteins. Here, to potentially discover novel entry inhibitors targeting CDV H, F and/or the cognate receptor: signaling lymphocyte activation molecule (SLAM) proteins, we designed a quantitative cell-based fusion assay that matched high-throughput screening (HTS) settings. By screening two libraries of small molecule compounds, we successfully identified two membrane fusion inhibitors (F2736-3056 and F2261-0043). Although both inhibitors exhibited similarities in structure and potency with the small molecule compound 3G (an AS-48 class morbilliviral F-protein inhibitor), F2736-3056 displayed improved efficacy in blocking fusion activity when a 3G-escape variant was employed. Altogether, we present a cell-based fusion assay that can be utilized not only to discover antiviral agents against CDV but also to dissect the mechanism of morbilliviral-mediated cell-binding and cell-to-cell fusion activity.

Pan-coronavirus fusion inhibitors possess potent inhibitory activity against HIV-1, HIV-2, and simian immunodeficiency virus

ABSTRACT EK1 peptide is a membrane fusion inhibitor with broad-spectrum activity against human coronaviruses (CoVs). In the outbreak of COVID-19, we generated a lipopeptide EK1V1 by modifying EK1 with cholesterol, which exhibited significantly improved antiviral activity. In this study, we surprisingly found that EK1V1 also displayed potent cross-inhibitory activities against divergent HIV-1, HIV-2, and simian immunodeficiency virus (SIV) isolates. Consistently, the recently reported EK1 derivative EK1C4 and SARS-CoV-2 derived fusion inhibitor lipopeptides (IPB02 ∼ IPB09) also inhibited HIV-1 Env-mediated cell–cell fusion and infection efficiently. In the inhibition of a panel of HIV-1 mutants resistant to HIV-1 fusion inhibitors, EK1V1 and IPB02-based inhibitors exhibited significantly decreased or increased activities, suggesting the heptad repeat-1 region (HR1) of HIV-1 gp41 being their target. Furthermore, the sequence alignment and molecular docking analyses verified the target site and revealed the mechanism underlying the resistance. Combined, we conclude that this serendipitous discovery provides a proof-of-concept for a common mechanism of viral fusion and critical information for the development of broad-spectrum antivirals.

Cholesterol: A key player in membrane fusion that modulates the efficacy of fusion inhibitor peptides.

The interaction of cholesterol with the neighboring lipids modulates several physical properties of the membrane. Mostly, it affects membrane fluidity, membrane permeability, lateral diffusion of lipids, bilayer thickness, and water penetration into the lipid bilayer. Due to the smaller head group to hydrophobic cross-sectional area of the tail, cholesterol induces intrinsic negative curvature to the membrane. The interaction of cholesterol with sphingolipids forms lipid rafts; generates phase separation in the membrane. The cholesterol-dependent modifications of membrane physical properties modulate viral infections by affecting the fusion between viral and host cell membranes. Cholesterol demonstrates a strong impact on the structure, depth of penetration, conformation, and organization of fusion peptides in membrane milieu. Further, cholesterol has been implicated to modify the fusion inhibitory efficiency of peptide-based membrane fusion inhibitors.

Triazavirin might be the new hope to fight Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Since the beginning of the outbreak, a large number of clinical trials have been registered worldwide, and thousands of drugs have been investigated to face new health emergency of highly contagious COVID-19 caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Drug repurposing, i.e., utilizing an approved drug for a different indication, offers a time- and cost-efficient alternative for making new (relevant) therapies available to physicians and patients. Considering given strategy, many approved and investigational antiviral compounds, alone or in various relevant combinations, used in the past to fight Severe Acute Respiratory Syndrome Coronavirus-1, Middle East Respiratory Syndrome Coronavirus, Human Immunodeficiency Virus type 1, or Influenza viruses are being evaluated against the SARS-CoV-2. Triazavirin (TZV), a non-toxic broad--spectrum antiviral compound, is efficient against various strains of the Influenza A virus (Influenza Virus A, Orthomyxoviridae), i.e., swine flu (H1N1, or H3N2), avian influenza (H5N1, H5N2, H9N2, or highly pathogenic H7N3 strain), Influenza B virus (Influenza Virus B, Orthomyxoviridae), Respiratory Syncytial Virus (Orthopneumovirus, Pneumoviridae), Tick-Borne Encephalitis Virus (known as Forest-Spring Encephalitis Virus; Flavivirus, Flaviviridae), West Nile Virus (Flavivirus, Flavaviridae), Rift Valley Fever Virus (Phlebovirus, Bunyaviridae), and Herpes viruses (Simplexviruses, Herpesviridae) as well. In regard to COVID-19, the molecule probably reduced inflammatory reactions, thus limiting the damage to vital organs and reducing the need for therapeutic support, respectively. In addition, in silico computational methods indicated relatively satisfactory binding affinities of the TZV ligand to both structural (E)- and (S)-proteins, non-structural 3-chymotrypsin-like protease (3-CLpro) of SARS-CoV-2 as well as human angiotensin-I converting enzyme-2 (ACE-2). The interactions between TZV and given viral structures or the ACE-2 receptor for SARS-CoV-2 might effectively block both the entry of the pathogen into a host cell and its replication. Promising treatment patterns of COVID-19 positive patients might be also based on a suitable combination of a membrane fusion inhibitor (umifenovir, for example) with viral RNA synthesis and replication inhibitor (TZV).

Unraveling the mechanism of arbidol binding and inhibition of SARS-CoV-2: Insights from atomistic simulations

The COVID-19 pandemic has spread rapidly and poses an unprecedented threat to the global economy and human health. Broad-spectrum antivirals are currently being administered to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). China's prevention and treatment guidelines suggest the use of an anti-influenza drug, arbidol, for the clinical treatment of COVID-19. Reports indicate that arbidol could neutralize SARS-CoV-2. Monotherapy with arbidol is superior to lopinavir-ritonavir or favipiravir for treating COVID-19. In SARS-CoV-2 infection, arbidol acts by interfering with viral binding to host cells. However, the detailed mechanism by which arbidol induces the inhibition of SARS-CoV-2 is not known. Here, we present atomistic insights into the mechanism underlying membrane fusion inhibition of SARS-CoV-2 by arbidol. Molecular dynamics (MD) simulation-based analyses demonstrate that arbidol binds and stabilizes at the receptor-binding domain (RBD)/ACE2 interface with a high affinity. It forms stronger intermolecular interactions with the RBD than ACE2. Analyses of the detailed decomposition of energy components and binding affinities revealed a substantial increase in the affinity between the RBD and ACE2 in the arbidol-bound RBD/ACE2 complex, suggesting that arbidol generates favorable interactions between them. Based on our MD simulation results, we propose that the binding of arbidol induces structural rigidity in the viral glycoprotein, thus restricting the conformational rearrangements associated with membrane fusion and virus entry. Furthermore, key residues of the RBD and ACE2 that interact with arbidol were identified, opening the door for developing therapeutic strategies and higher-efficacy arbidol derivatives or lead drug candidates.

The Antimalarial Compound Atovaquone Inhibits Zika and Dengue Virus Infection by Blocking E Protein-Mediated Membrane Fusion.

Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6–2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.

Scaffold morphing of arbidol (umifenovir) in search of multi-targeting therapy halting the interaction of SARS-CoV-2 with ACE2 and other proteases involved in COVID-19

The rapid emergence of novel coronavirus, SARS-coronavirus 2 (SARS-CoV-2), originated from Wuhan, China, imposed a global health emergency. Angiotensin-converting enzyme 2 (ACE2) receptor serves as an entry point for this deadly virus while the proteases like furin, transmembrane protease serine 2 (TMPRSS2) and 3 chymotrypsin-like protease (3CLpro) are involved in the further processing and replication of SARS-CoV-2. The interaction of SP with ACE2 and these proteases results in the SARS-CoV-2 invasion and fast epidemic spread. The small molecular inhibitors are reported to limit the interaction of SP with ACE2 and other proteases. Arbidol, a membrane fusion inhibitor approved for influenza virus is currently undergoing clinical trials against COVID-19. In this context, we report some analogues of arbidol designed by scaffold morphing and structure-based designing approaches with a superior therapeutic profile. The representative compounds A_BR4, A_BR9, A_BR18, A_BR22 and A_BR28 restricted the interaction of SARS-CoV-2 SP with ACE2 and host proteases furin and TMPRSS2. For 3CLPro, Compounds A_BR5, A_BR6, A_BR9 and A_BR18 exhibited high binding affinity, docking score and key residue interactions. Overall, A_BR18 and A_BR28 demonstrated multi-targeting potential against all the targets. Among these top-scoring molecules A_BR9, A_BR18, A_BR22 and A_BR28 were predicted to confer favorable ADME properties.

The N-Terminal Region of Soybean PM1 Protein Protects Liposomes during Freeze-Thaw

Late embryogenesis abundant (LEA) group 1 (LEA_1) proteins are intrinsically disordered proteins (IDPs) that play important roles in protecting plants from abiotic stress. Their protective function, at a molecular level, has not yet been fully elucidated, but several studies suggest their involvement in membrane stabilization under stress conditions. In this paper, the soybean LEA_1 protein PM1 and its truncated forms (PM1-N: N-terminal half; PM1-C: C-terminal half) were tested for the ability to protect liposomes against damage induced by freeze-thaw stress. Turbidity measurement and light microscopy showed that full-length PM1 and PM1-N, but not PM1-C, can prevent freeze-thaw-induced aggregation of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes and native thylakoid membranes, isolated from spinach leaves (Spinacia oleracea). Particle size distribution analysis by dynamic light scattering (DLS) further confirmed that PM1 and PM1-N can prevent liposome aggregation during freeze-thaw. Furthermore, PM1 or PM1-N could significantly inhibit membrane fusion of liposomes, but not reduce the leakage of their contents following freezing stress. The results of proteolytic digestion and circular dichroism experiments suggest that PM1 and PM1-N proteins bind mainly on the surface of the POPC liposome. We propose that, through its N-terminal region, PM1 functions as a membrane-stabilizing protein during abiotic stress, and might inhibit membrane fusion and aggregation of vesicles or other endomembrane structures within the plant cell.

Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance.IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

Preparation and evaluation of amphipathic lipopeptide-loaded PLGA microspheres as sustained-release system for AIDS prevention.

At present, AIDS drugs are typical inhibitors that cannot achieve permanent effects. Therefore, the research of blocking HIV infection is essential. Especially for people in the high‐risk environment, long‐term prevention is important, because HIV can easily infect cells once the drug is interrupted. However, there is still no long‐acting AIDS prevention drug approved. Hence, the purpose of this study is to prepare a fusion inhibitor loaded poly(d, l‐lactic‐co‐glycolic acid) (PLGA) microspheres as a sustained‐release system for long‐term AIDS prevention. As the HIV membrane fusion inhibitor (LP‐98) used in this research is amphiphilic lipopeptide, W1/O/W2 double‐emulsion method was chosen, and premix membrane emulsification technique was used for controlling the uniformity of particle size. Several process parameters that can impact drug loading efficiency were summarized: the concentration of LP‐98 and PLGA, and the preparation condition of primary emulsion. Finally, the microspheres with high loading efficiency (>8%) and encapsulation efficiency (>90%) were successfully prepared under optimum conditions. Pharmacokinetic studies showed that LP‐98‐loaded microspheres were capable to continuously release for 24 days in rats. This research can promote the application of sustained‐release microspheres in AIDS prevention, and the embedding technique used in this study can also provide references for the loading of other amphipathic drugs.

Miltefosine inhibits the membrane remodeling caused by phospholipase action by changing membrane physical properties

Miltefosine (hexadecylphosphocholine or HePC) is an alkylphosphocholine approved for the treatment of visceral and cutaneous Leishmaniasis. HePC exerts its effect by interacting with lipid membranes and affecting membrane-dependent processes. The molecular geometry of HePC suggests that the pharmacological function of HePC is to alter membrane curvature. As a model system, we studied the enzyme production in model membranes of diacylglycerol (DAG) or ceramide (CER), lipids involved in cell signaling which alter the structure of membranes. Here, we studied the effect of HePC on changes in phospholipase activity and on the effect that the lipid products have on the curvature and fusogenicity of membranes where they accumulate. Our results indicate that HePC inhibits the long-time restructuring of membranes, characteristic of the DAG and CER enzyme formation processes. In addition, the drug also reduces the fusogenicity of phospholipase-derived products. We postulate that the effect of HePC is due to a non-specific geometric compensation of HePC to the inverted cone-shape of DAG and CER products, acting as a relaxation agent of membrane curvature stress. These data are important for understanding the mechanism of action by which HePC regulates the lipid metabolism and signal transduction pathways in which these enzymes are involved.

Design of Potent Membrane Fusion Inhibitors against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity.

The 2019 coronavirus disease (COVID-19), caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed serious threats to global public health and economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we first verified that SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as a cell receptor and that its spike (S) protein mediates high membrane fusion activity. The heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR2) site, than the HR1 sequence in S2 of severe acute respiratory syndrome coronavirus (SARS-CoV). Then, we designed an HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly potent activities in inhibiting SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the structure-activity relationship (SAR) of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the results presented here provide important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. The S protein of coronaviruses mediates viral receptor binding and membrane fusion, thus being considered a critical target for antivirals. Herein, we report that the SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S protein of SARS-CoV that emerged previously. The HR1 sequence in the fusion protein of SARS-CoV-2 adopts a much higher helical stability than the HR1 sequence in the fusion protein of SARS-CoV and can interact with the HR2 site to form a six-helical bundle structure more efficiently, underlying the mechanism of the enhanced fusion capacity. Also, importantly, the design of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and tools to exploit the fusion mechanism.

Experimental Evolution To Isolate Vaccinia Virus Adaptive G9 Mutants That Overcome Membrane Fusion Inhibition via the Vaccinia Virus A56/K2 Protein Complex.

For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the ΔA56 virus, the G9H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter's G9 subcomponent. Although the G9H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.

Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors.

Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

Small Molecule Inhibition of Membrane Fusion Mediated by the Flavivirus Envelope Protein

Despite the successes of antiviral therapies for the human immunodeficiency virus and hepatitis C virus, the number of validated antiviral targets is quite small and combination therapy remains the only effective strategy for avoiding viral resistance against these and other RNA viruses. Despite the success of the humoral immune system in targeting viral envelope (E) proteins, they remain a challenging target for rational inhibitor design efforts. E proteins act as catalysts lowering the energy barrier for fusion of the viral membrane with specific cellular membranes during viral entry. Unlike classical enzymes, however, E proteins lack a discrete active site where specific covalent bonds are made or broken; moreover, the chemical reaction they catalyze is not amenable to mimicry by small molecule transition state analogs or to other rational approaches of inhibitor design. We have used phenotypic screening, medicinal chemistry, and chemical biology to investigate pharmacological inhibition of the dengue virus envelope protein, E, and to develop tools that enable the discovery and optimization of direct-acting antivirals that act against this non-traditional target.

SARS-Cov-2, The Covid-19 Pandemic Reminds us of the Antiviral Properties of Chlorpromazine

In vitro studies have established the classic antipsychotic drug chlorpromazine (CPZ) as an effective antiviral agent against various viruses, including corona viruses [1]. Also, SARS-CoV-2, the cause of the current Covid 19 pandemic, is inhibited by CPZ [2]. CPZ is known to act on clathrin-mediated endocytosis (CME), which affects the formation of clathrin-coated pits on the plasma membrane [3]. This pathway is one of the most important entry routes of viruses, more precisely virions, into host cells. Studies on the antiviral effect of CPZ have shown that it influences different mechanisms of virus formation, including post-entry effects. CPZ belongs to the group of cationic amphiphilic drugs (CADs), which have the propensity to interact with various cell structures, especially membranes. They accumulate in intracellular compartments (acidic), such as early and late endosomes/lysosomes crucial for virus formation (e.g. particle coating, assembly or budding) [4]. In the case of corona viruses (SARS-CoV), CPZ inhibits the cell entry chiefly by CME [5]. However, CPZ it is also known to inhibit membrane fusion processes at the plasma membrane or at an intracellular location following virus uptake by endocytosis. Among them, CME and cathepsin mediated S protein cleavage are two critical steps for viral entry and infection of corona viruses (all coronaviruses are enveloped viruses with a long single-plus stranded RNA) [6]. Notably, SARS-CoV and SARS-CoV-2 use exactly the same attachment receptor (ACE2) and serine protease (TMPRSS2/S protein priming) for cell entry [7]. However, the exact nature of the viral entry is context-dependent, including the type of the virus and the type of the host cells. As mentioned above, SARS-CoV-2 utilizes the ACE2-receptor (angiotensin converting enzyme II) for viral entry into the host cells. ACE2 transcripts was originally only found in heart, kidney and testis of humans. However, it was later found that ACE2 protein expresses abundantly in the epithelia of the human lung, small intestine and brain [8].

Cholesterol alters the inhibitory efficiency of peptide-based membrane fusion inhibitor

The membrane composition modulates membrane fusion by altering membrane physical properties and the structure, organization and dynamics of fusion proteins and peptides. The journey of developing peptide-based viral fusion inhibitors is often stalled by the change in lipid composition of viral and target membranes. This makes it important to study the role of membrane composition on the organization, dynamics and fusion inhibiting abilities of the peptide-based fusion inhibitors. Cholesterol, an important constituent of mammalian cell membrane, modulates bilayer properties in multiple ways and impart its effect on the membrane fusion. We have previously shown that TG-23 peptide derived from phagosomal coat protein, coronin 1, shows significant inhibition of fusion between membranes without cholesterol. In this work, we have studied the effect of the TG-23 peptide on the polyethylene glycol-mediated membrane fusion in presence of different concentrations of membrane cholesterol. Our results show that the inhibitory effect of TG-23 is being completely reversed in cholesterol containing membranes. We have evaluated the structure, organization, dynamics and depth of penetration of TG-23 in membranes having different lipid compositions and its effect on membrane properties. Our results demonstrate that cholesterol does not affect the secondary structure of the peptide, however, alters the depth of penetration of the peptide and modifies peptide organization and dynamics. The cholesterol dependent change in organization and dynamics of the peptide influences its efficacy in membrane fusion. Therefore, we envisage that the study of peptide organization and dynamics is extremely important to determine the effect of peptide on the membrane fusion.

From the Editor’s desk…: June 2019

From the Editor’s desk…: June 2019