A highly potent and stable pan-coronavirus fusion inhibitor as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases

The development of broad-spectrum antivirals against human coronaviruses (HCoVs) is critical to combat the current coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, as well as future outbreaks of emerging CoVs. We have previously identified a polyethylene glycol-conjugated (PEGylated) lipopeptide, EK1C4, with potent pan-CoV fusion inhibitory activity. However, PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo. Therefore, we herein report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment (HR2-CF) of HCoV-OC43. Among these lipopeptides, EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike (S) mutants, as well as other HCoVs and some bat SARS-related coronaviruses (SARSr-CoVs) tested. The dePEGylated lipopeptide EKL1C exhibited significantly stronger resistance to proteolytic enzymes, better metabolic stability in mouse serum, higher thermostability than the PEGylated lipopeptide EK1C4, suggesting that EKL1C could be further developed as a candidate prophylactic and therapeutic for COVID-19 and other coronavirus diseases.

Identification of the dietary supplement capsaicin as an inhibitor of Lassa virus entry

The limited treatment options for the increasing occurrence of Lassa hemorrhagic fever in West Africa poses an urgent need for the discovery and development of novel therapeutics. Dietary supplements, especially natural products that are edible and safe for human use, are a good source of drug discovery with potential for uncovering novel applications. In this study, we tested 40 natural products of dietary supplements and identified capsaicin, a common dietary supplement abundant in chili peppers, as an inhibitor of Lassa virus (LASV) entry with EC of 6.9-10.0 μmol/L using an HIV based pseudovirus platform. Capsaicin inhibits the entry of five LASV strains but not against the Old World arenavirus lymphocytic choriomeningitis virus (LCMV), showing a preferential activity against LASV. Capsaicin inhibits LASV entry by blocking the pH dependent viral fusion through affecting the stable signal peptide (SSP)-GP2 transmembrane (GP2) region of the LASV surface glycoprotein. Mutational study revealed the key residues Ala25, Val431, Phe434 and Val435 in SSP-GP2 region in capsaicin's antiviral effect. This study for the first time reveals a direct acting antiviral effect of capsaicin against the hemorrhagic fever causing LASV, providing detailed interaction hot spots in the unique SSP-GP2 interface of LASV glycoprotein that is crucial in fusion inhibition, and offering a new strategy in discovering and developing antivirals from natural products that are safe for human use.

Characterization of Gp41 Polymorphisms in the Fusion Peptide Domain and T-20 (Enfuvirtide) Resistance-Associated Regions in Korean HIV-1 Isolates

HIV-1 gp41 is an envelope protein that plays an essential role in virus entry. The mutation of gp41 affects HIV-1 entry and susceptibility to the fusion inhibitor T-20. Therefore, we analyzed the natural polymorphism of gp41 of 163 HIV-1 isolates from T-20-naive Koreans infected with HIV-1. This study of gp41 polymorphisms showed that insertions in the fourth threonine (74.8%) and L7M substitutions (85.3%) were more frequent in the fusion peptide motif in Korean HIV-1 isolates compared with those from other countries. Minor T-20 resistance mutations such as L45M (1.2%), N126K (1.2%), and E137K (6.7%) were detected, but the critical T-20 resistance mutations were not detected in the gp41 HR1 and HR2 regions. In addition, the N42S mutation (12.9%) associated with T-20 hypersusceptibility was detected at a high frequency. These results may serve as useful data for studies considering T-20 for use in the development of a more effective anti-retroviral treatment in Korea.

Strategy for Developing Medical Arsenals by Modulation of Membrane Fusion Activity of Influenza Virus Hemagglutinin

Influenza virus is a serious pathogen that burdens society with health care costs, and can lead to fatality. The virus is dealt with currently by vaccination and anti-influenza drugs. However, vaccines need to be improved towards safer and more efficient production formats, and drugs need to be constantly renewed to cope with resistances. That the neuraminidase inhibitors are only drugs currently available warrants urgent attention to an alternative anti-influenza target. In this paper we introduce studies on fusion activity of influenza virus hemagglutinin (HA), and discuss how to best utilize the knowledge for an improved vaccine development and an anti-influenza drug search. Potential application of mutations resulting in changes in fusion activity to cell culture optimized vaccine virus development and strategies to develop broad spectrum anti-influenza drugs through targeting the conserved fusion domain of the HA are discussed.

Enfuvirtide: el primer paso de una nueva estrategia de tratamiento antirretroviral / Enfuvirtide: the first step for a new strategy of antiretroviral therapy

Enfuvirtide (antes T-20) es el primer inhibidor de la entrada a la célula del HIV-1 en ser aprobado. Es un péptido análogo de la porción HR2 de la glucoproteína de superficie viral gp41. Su mecanismo de acción consiste en la unión competitiva a la porción HR1 de la gp41 para impedir los cambios conformacionales del complejo gp41-gp120 tras la unión del HIV-1 a los receptores celulares, impidiendo así el acercamiento y posterior fusión entre el virus y la célula. Se aplica por vía subcutánea. Los resultados de los principales estudios clínicos (TORO 1 y 2) llevados a cabo en pacientes con fallo virológico, tratamientos previos con antirretrovirales y portadores de cepas virales altamente resistentes, mostraron que quienes recibieron enfuvirtide + HAART optimizado, elegido mediante un test de resistencia, presentaron mayores beneficios que quienes sólo recibieron HAART optimizado, en términos de mejor recuperación inmune y mayor descenso de la carga viral de HIV. Los mejores resultados se observaron en el subgrupo de pacientes con más drogas útiles en el HAART según el test de resistencia, una menor carga viral de HIV y un mayor recuento de linfocitos CD4 basales. El principal efecto adverso es el desarrollo de lesiones por hipersensibilidad en los sitios de aplicación. El alto costo de enfuvirtide se vio compensado por una reducción en los costos de internación.

Enfuvirtide (T-20) is the first approved HIV-1 entry into cells' inhibitor. It is a peptide with an amino acid sequence analogue to HR2 region of the viral surface glycoprotein gp41. Its mechanism of action is the competitive binding to HR1 region of the gp41, preventing the interaction between HR1 and HR2 and impeding the conformational changes in gp41 necessary for fusion of the virus with the cell. Its application is by subcutaneous injection. The main clinical trials of enfuvirtide (TORO 1 and 2) were performed in HIV-infected patients with virological failure, high antiretroviral experience and highly resistant viral isolates. Those trials showed that the addition of enfuvirtide to an optimized HAART (chosen with a resistance test) provided better results than HAART alone, measured by drop in viral load and immunologic benefit. The best results were observed in the subgroup of patients with more useful drugs in HAART (according to the information of the resistance test), a lower viral load, and a higher CD4 cell count at baseline. The most important adverse event is the production of injection drug hypersensitivity reaction in 98% of patients. The high cost is compensated by a reduction in costs derived from admissions.

A prevalência de fatores de risco para baixa adesão na terapia com enfuvirtida, nos usuários soropositivos para o HIV em tratamento nos centros de referência em Porto Alegre - RS / The prevalence of risk factors for decreasing adhesion in the therapy with enfuvirtida in HIV infected users in treatment in centers of reference in Porto Alegre - RS

Introdução: Em 2005 o Ministério da Saúde do Brasil disponibilizou a enfuvirtida, o primeiro inibidor de fusão, uma nova classe de anti-retrovirais com ação extracelular. As dificuldades relatadas pelos primeiros usuários, em um dos centros de referência, e a carência de estudos envolvendo os usuários do Sistema Único de Saúde motivaram esta pesquisa. Objetivo: identificar os fatores de risco para baixa adesão ao tratamento com Enfuvirtida. Métodos: foram entrevistadas 37 pessoas de Porto Alegre, compreendendo a população total de usuários de enfuvirtida nesta cidade até o mês de outubro de 2006. Resultados: 27% dos pacientes já pensaram em desistir do tratamento, 22,2% referiram esquecimento de dose nos últimos 30 dias e 45,9% não souberam diferenciar HIV de aids. Os efeitos adversos locais ocorreram em todos os usuários, houve dor nos locais de aplicação em 81,1% dos casos, fato que não atrapalhou as atividades diárias (70,3%). Quanto às aplicações, 28,9% não tiveram a primeira dose supervisionada, 27% não realizaram massagem após as aplicações, 46% dos pacientes usavam regiões não orientadas pela enfermagem para aplicação, 29,7% acharam “difícil” encontrar um local para aplicar. Conclusão: o estudo reforça a necessidade do acompanhamento direto por um profissional de enfermagem para aplicação do medicamento, visto que os procedimentos podem parecer fáceis, mas requerem boa técnica para diluição, aspiração e aplicação a fim de diminuir os efeitos adversos locais, assim como as orientações sobre os cuidados após a aplicação.

Introduction: In 2005 the Ministry Health of Brazil provided Enfuvirtide, the first inhibitor of fusion, a new class of anti-retroviraes with extracellular action. The difficulties described by the first users, in one of the reference centers, and the lack of studies with the users of the Public Health Department, had motivated this research. Objective: to identify risk factors for low adhesion to the treatment with enfuvirtida. Methods: 37 people from Porto Alegre had been interviewed, the total population of users of enfuvirtide in this city until October, 2006. Results: had presented that 27% of the patients had already thought about giving up the treatment, 22,2% had forgotten to take the dose in the last 30 days and 45,9% do not know the difference between HIV and AIDS. The local adverse effect had occurred in all the users, pain in the application places were present in 81,1% of the cases, but it did not interfere in daily activities (70,3%). Concerning applications, 28,9% had not had a first supervised dose, 27% did not make a massage after the applications, 46% of the patients also used regions for application which were not guided by the nurses, 29,7% had had “difficulties” in finding a place to apply. Conclusion: the study strengthens the necessity of the direct accompaniment by a nursing professional, although the procedures can seem easy, they require good technique for dilution, aspiration and application to diminish the local adverse effects, as well as the orientation on the care after the application.