HIV-1 Integrase Tetramers Are the Antiviral Target of Pyridine-Based

RESEARCH ARTICLE HIV-1 integrase tetramers are the antiviral target of pyridine-based allosteric integrase inhibitors Pratibha C Koneru1, Ashwanth C Francis2, Nanjie Deng3, Stephanie V Rebensburg1, Ashley C Hoyte1, Jared Lindenberger1, Daniel Adu-Ampratwum4, Ross C Larue4, Michael F Wempe5, Alan N Engelman6,7, Dmitry Lyumkis8, James R Fuchs4, Ronald M Levy9, Gregory B Melikyan2, Mamuka Kvaratskhelia1* 1Division of Infectious Diseases, School of Medicine, University of Colorado, Aurora, United States; 2Division of Infectious Diseases, Department of Pediatrics, Emory University, Atlanta, United States; 3Department of Chemistry and Physical Sciences, Pace University, New York, United States; 4College of Pharmacy, The Ohio State University, Columbus, United States; 5Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver, Aurora, United States; 6Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, United States; 7Department of Medicine, Harvard Medical School, Boston, United States; 8Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, United States; 9Department of Chemistry, Temple University, Philadelphia, United States Abstract Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising new class of antiretroviral agents that disrupt proper viral maturation by inducing hyper-multimerization of IN. Here we show that lead pyridine-based ALLINI KF116 exhibits striking selectivity for IN tetramers *For correspondence: versus lower order protein oligomers. IN structural features that are essential for its functional mamuka.kvaratskhelia@ucdenver. tetramerization and HIV-1 replication are also critically important for KF116 mediated higher-order edu IN multimerization. Live cell imaging of single viral particles revealed that KF116 treatment during Competing interest: See virion production compromises the tight association of IN with capsid cores during subsequent page 22 infection of target cells. We have synthesized the highly active (-)-KF116 enantiomer, which Funding: See page 22 displayed EC50 of ~7 nM against wild type HIV-1 and ~10 fold higher, sub-nM activity against a clinically relevant dolutegravir resistant mutant virus suggesting potential clinical benefits for Received: 23 February 2019 complementing dolutegravir therapy with pyridine-based ALLINIs. Accepted: 22 May 2019 Published: 23 May 2019 Reviewing editor: Julie Overbaugh, Fred Hutchinson Cancer Research Center, United Introduction States Multifunctional HIV-1 integrase (IN) is an important therapeutic target. HIV-1 IN strand transfer inhibitors (INSTIs), which have become a first-line therapy to treat HIV-1 infected patients, block the cata- Copyright Koneru et al. This lytic function of the viral protein during the early phase of infection and thus prevent integration of article is distributed under the viral cDNA into human chromosomes (Hazuda, 2012; Hazuda et al., 2000; McColl and Chen, terms of the Creative Commons Attribution License, which 2010). Clinical applications of first generation INSTIs raltegravir (RAL) and elvitegravir (EVG) resulted permits unrestricted use and in evolution of drug resistant phenotypes containing IN substitutions in the vicinity of the inhibitor redistribution provided that the binding sites (Cooper et al., 2008; Garrido et al., 2012). Second generation dolutegravir (DTG), original author and source are bictegravir (BIC) and investigational drug cabotegravir (CAB) exhibit significantly higher genetic credited. pressure for the evolution of drug resistance and yield complex resistance pathways with IN Koneru et al. eLife 2019;8:e46344. DOI: https://doi.org/10.7554/eLife.46344 1 of 27 Research article Microbiology and Infectious Disease eLife digest HIV-1 inserts its genetic code into human genomes, turning healthy cells into virus factories. To do this, the virus uses an enzyme called integrase. Front-line treatments against HIV-1 called “integrase strand-transfer inhibitors” stop this enzyme from working. These inhibitors have helped to revolutionize the treatment of HIV/AIDS by protecting the cells from new infections. But, the emergence of drug resistance remains a serious problem. As the virus evolves, it changes the shape of its integrase protein, substantially reducing the effectiveness of the current therapies. One way to overcome this problem is to develop other therapies that can kill the drug resistant viruses by targeting different parts of the integrase protein. It should be much harder for the virus to evolve the right combination of changes to escape two or more treatments at once. A promising class of new compounds are “allosteric integrase inhibitors”. These chemical compounds target a part of the integrase enzyme that the other treatments do not yet reach. Rather than stopping the integrase enzyme from inserting the viral code into the human genome, the new inhibitors make integrase proteins clump together and prevent the formation of infectious viruses. At the moment, these compounds are still experimental. Before they are ready for use in people, researchers need to better understand how they work, and there are several open questions to answer. Integrase proteins work in groups of four and it is not clear how the new compounds make the integrases form large clumps, or what this does to the virus. Understanding this should allow scientists to develop improved versions of the drugs. To answer these questions, Koneru et al. first examined two of the new compounds. A combination of molecular analysis and computer modelling revealed how they work. The compounds link many separate groups of four integrases with each other to form larger and larger clumps, essentially a snowball effect. Live images of infected cells showed that the clumps of integrase get stuck outside of the virus’s protective casing. This leaves them exposed, allowing the cell to destroy the integrase enzymes. Koneru et al. also made a new compound, called (-)-KF116. Not only was this compound able to tackle normal HIV-1, it could block viruses resistant to the other type of integrase treatment. In fact, in laboratory tests, it was 10 times more powerful against these resistant viruses. Together, these findings help to explain how allosteric integrase inhibitors work, taking scientists a step closer to bringing them into the clinic. In the future, new versions of the compounds, like (-)-KF116, could help to tackle drug resistance in HIV-1. substitutions in both the proximity of and significantly distanced from inhibitor binding sites (Cahn et al., 2013; Eron et al., 2013; Malet et al., 2018; Margolis et al., 2015; Smith et al., 2018; Tsiang et al., 2016). More recently, allosteric HIV-1 integrase (IN) inhibitors (ALLINIs; also referred to as non-catalytic site integrase inhibitors (NCINIs); LEDGINs or INLAIs), which are mechanistically distinct from INSTIs have been developed (Christ et al., 2010; Fader et al., 2014b; Le Rouzic et al., 2013; Sharma et al., 2014; Tsiang et al., 2012). Unlike INSTIs, ALLINIs are highly potent during virion maturation. They induce higher-order IN multimerization in virions and consequently inhibit IN-RNA interactions, which in turn yields eccentric noninfectious virions with the ribonucleoprotein com- plexes (RNPs) mislocalized outside of the translucent capsid (CA) cores (Balakrishnan et al., 2013; Desimmie et al., 2013; Fontana et al., 2015; Gupta et al., 2014; Jurado et al., 2013; Kessl et al., 2016; van Bel et al., 2014). ALLINIs also display a secondary, albeit significantly reduced, activity during early steps of HIV-1 infection where these compounds interfere with HIV-1 IN binding to its cognate cellular cofactor LEDGF/p75 (Christ and Debyser, 2013; Christ et al., 2012; Christ et al., 2010; Kessl et al., 2012; Le Rouzic et al., 2013; Sharma et al., 2014; Tsiang et al., 2012). The key pharmacophore in all potent ALLINIs includes both a carboxylic acid and tert-butoxy group, which extend from a core aromatic ring system (Figure 1A)(Christ et al., 2010; Jurado and Engelman, 2013; Sharma et al., 2014; Tsiang et al., 2012). The original series of compounds con- tained a quinoline core, exhibited relatively modest potency, and comparatively low genetic barrier to drug resistance (Christ et al., 2010; Fader et al., 2014b; Feng et al., 2013; Tsiang et al., 2012). Follow up SAR studies have extended in two directions: i) efforts that retain the quinoline core while Koneru et al. eLife 2019;8:e46344. DOI: https://doi.org/10.7554/eLife.46344 2 of 27 Research article Microbiology and Infectious Disease Figure 1. IN tetramers are preferentially targeted by pyridine and quinoline based ALLINIs. (A) Chemical structures of pyridine-based KF116 and quinoline-based BI224436. (B) SEC based separation of IN tetramer(T), dimer(D) and monomer(M) fractions. C and D, DLS analysis of 200 nM IN fractions in the presence of 1 mM KF116 (blue lines, (C) or BI224436 (red lines, (D). DMSO controls for each DLS experiment (C and D) are shown in gray. Representative results of three independent experiments at 30 mins time point are shown. The online version of this article includes the following figure supplement(s) for figure 1: Figure supplement 1. Pyridine and quinoline based ALLINIs preferentially target IN tetramers. varying the substituted groups have resulted in highly potent inhibitors such as BI224436 with antiviral EC50 of ~14 nM (Fader et al., 2014b)(Figure 1A); ii) other studies successfully explored different core ring structures

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