Characterization of New Cationic N,N‑Dimethyl[70]fulleropyrrolidinium Iodide Derivatives as Potent HIV‑1 Maturation Inhibitors Edison Castro, † Zachary S. Martinez, ‡ Chang-Soo Seong, ‡ Andrea Cabrera-Espinoza, † Mauro Ruiz, † Andrea Hernandez Garcia, † Federico Valdez, ‡ Manuel Llano,* ,‡ and Luis Echegoyen* ,† † Department of Chemistry, University of Texas at El Paso, 500 West University Avenue, CCSB #3.0302, El Paso, Texas 79968, United States ‡ Department of Biological Sciences, University of Texas at El Paso, 500 West University Avenue, El Paso, Texas 79968, United States * S Supporting Information ABSTRACT: HIV-1 maturation can be impaired by altering protease (PR) activity, the structure of the Gag-Pol substrate, or the molecular interactions of viral structural proteins. Here we report the synthesis and characterization of new cationic N,N- dimethyl[70]fulleropyrrolidinium iodide derivatives that inhibit more than 99% of HIV-1 infectivity at low micromolar concentrations. Analysis of the HIV-1 life cycle indicated that these compounds inhibit viral maturation by impairing Gag and Gag-Pol processing. Importantly, fullerene derivatives 2a-c did not inhibit in vitro PR activity and strongly interacted with HIV immature capsid protein in pull-down experiments. Furthermore, these compounds potently blocked infectivity of viruses harboring mutant PR that are resistant to multiple PR inhibitors or mutant Gag proteins that confer resistance to the maturation inhibitor Bevirimat. Collectively, our studies indicate fullerene derivatives 2a-c as potent and novel HIV-1 maturation inhibitors. ■ INTRODUCTION The emergence of resistant human immunodeficiency virus (HIV) strains limits the therapeutic efficiency of current antiretroviral therapies. 1 Therefore, discovery of new antiviral agents remains an important goal for HIV-1 infection treatment. These agents may act by impairing viral maturation, a process in which Gag and Gag-Pol polyproteins are sequentially cleaved by PR to produce viral enzymes and structural proteins that are required for viral replication. 2 Pharmacological and genetic evidence demonstrates that impairment of viral maturation is a powerful strategy to block HIV-1 replication in vivo and in vitro. HIV-1 is released from infected cells in the form of immature, noninfectious virions that must undergo maturation before acquiring full infectivity. Viral maturation is triggered by proteolytic processing of Gag and Gag-Pol polyproteins by HIV-1 PR. This processing results in the production of functional viral proteins including capsid (CA) proteins, which assemble into the viral core. HIV-1 maturation can be hindered by drugs that act as PR inhibitors (PIs) or as maturation inhibitors (MIs). The latter can bind to Gag and affect its processing or to CA, thus impairing core assembly. MIs binding to mature CA do not affect PR-mediated processing, whereas those targeting CA-SP1 selectively block the cleavage between CA and SP1, allowing normal processing of Gag and Gag-Pol at other cleavage sites. 3 Currently, there are no maturation inhibitors used clinically. 4 Since the discovery of fullerene C 60 , 5 efficient synthetic methods for fullerene functionalization have been developed. 6 Functionalization with highly polar or ionic groups is the most commonly used approach to obtain water-soluble fullerene derivatives for biomedical applications. 7 C 60 and C 70 fullerene derivatives have been shown to affect HIV-1 replication. 8 The currently accepted fullerene-induced inhibition mechanism suggests binding to the PR active site 9 as was determined by analysis of the effect of these compounds on the in vitro activity Received: July 7, 2016 Published: November 17, 2016 Article pubs.acs.org/jmc © 2016 American Chemical Society 10963 DOI: 10.1021/acs.jmedchem.6b00994 J. Med. Chem. 2016, 59, 10963-10973