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Discovery of a Distinct Chemical and Mechanistic Class of Allosteric HIV-1 Integrase Inhibitors with Antiretroviral Activity Christine Burlein, Cheng Wang, Xu Min, Triveni Bhatt, Mark Stahlhut, Yangsi Ou, Gregory C Adam, Jeffrey Heath, Daniel J. Klein, John Sanders, Kartik Narayan, Pravien Abeywickrema, Mee Ra Heo, Steven S. Carroll, Jay A. Grobler, Sujata Sharma, Tracy L Diamond, Antonella Converso, and Daniel J. Krosky ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00550 • Publication Date (Web): 12 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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ACS Chemical Biology

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Discovery of a Distinct Chemical and Mechanistic Class of Allosteric HIV-1 Integrase

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Inhibitors with Antiretroviral Activity

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Authors: Christine Burlein†, Cheng Wang‡, Min Xu†, Triveni Bhatt†, Mark Stahlhut†, Yangsi

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Ou†, Gregory C. Adamǁ, Jeffrey Heathǁ, Daniel J. Klein#, John Sanders#, Kartik Narayan†,

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Pravien Abeywickremaǁ, Mee Ra Heoǁ, Steven S. Carroll†, Jay A. Grobler§, Sujata Sharmaǁ,

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Tracy L. Diamond§, Antonella Converso‡ and Daniel J. Krosky†*

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†

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and §Infectious Disease Biology, Merck & Co., Inc., West Point, PA, 19486, USA.

Pharmacology, ‡Discovery Chemistry, ǁScreening and Protein Science, #Structural Chemistry,

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ABSTRACT: Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-

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derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral

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effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes

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that have catastrophic effects on viral maturation, rendering the virions assembled in the

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presence of ALLINIs non-infectious. A high-throughput screen for compounds that disrupt the

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IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-

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butylsulfonamide series, as exemplified by 1. The chemical, biochemical and virological

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characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype

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through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target

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engagement and differentiating this new series from the existing ALLINIs was the development

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of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series.

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These findings further solidify the late antiviral of mechanism of ALLINIs, and point towards

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opportunities to develop structurally and mechanistically novel antiretroviral agents with unique

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resistance patterns.

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INTRODUCTION:

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Since its identification over thirty years ago, HIV/AIDS remains a global scourge, with

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~37 million people infected with HIV worldwide1. The implementation of highly active

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antiretroviral therapy (HAART) in the 1990’s has had a dramatic effect on patient health through

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suppression of viral replication, increasing CD4+ T cell levels, decreasing mortality rates and

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lowering the rates of viral transmission2-6. However, patients on HAART must remain on these

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antiretroviral drugs for life to prevent viral rebound7. Due to the emergence of drug resistant

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virus, tolerability issues, and potential drug-drug interactions, there is a continued need for the

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development of new antiviral agents8.

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HIV-1 integrase (IN), which catalyzes the integration of proviral DNA into the host

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genome, has been an attractive target for antiretroviral drug development9. Raltegravir, the first

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FDA-approved HIV integrase strand-transfer inhibitor (INSTI)10, binds in the active site to the

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IN·proviral DNA complex following 3’-processing, trapping the pre-integration complex11-13. In

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contrast, allosteric integrase inhibitors (ALLINIs) are a newer class of compounds that bind to

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the lens epithelial-derived growth factor (LEDGF) binding site located in the IN catalytic core

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domain (CCD)14. LEDGF is a host chromatin factor containing both DNA- and protein-protein

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interaction domains, and is associated with regions undergoing active transcription15-17. The

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interaction of the IN CCD to LEDGF serves to localize the viral intasome to these actively

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transcribed genomic regions, facilitating successful proviral DNA integration and improving the

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production of retroviral RNA to levels that support viral propagation18-22.


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ALLINIs, discovered independently by both high-throughput screening (HTS)23 and

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structure-based methods14, contain a critical t-butoxyacid pharmacophore that mimics key

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interactions made by LEDGF to the CCD. ALLINIs have been optimized to have potent

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antiviral activity and drug-like properties, and are not affected by INSTI resistance mutations24.

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However, as the mechanisms of action of ALLINIs were characterized, it became clear that their

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engagement to the LEDGF-binding pocket have multimodal effects on both IN function and the

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retroviral lifecycle25-27. In addition to being protein-protein interaction inhibitors, the binding of

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ALLINIs to the LEDGF pocket triggers multimerization of IN, both in vitro and in cellulo,

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forming high molecular weight aggregates28-30. The ability of IN to form enzymatically

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productive complexes with proviral and host DNA is dependent on its conformational and

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oligomeric dynamics31 and accordingly, ALLINIs are potent LEDGF-independent inhibitors in

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biochemical integration assays25, 26.

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Despite the ability of ALLINIs to block multiple IN functions critical for proviral DNA

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integration, these compounds are substantially more potent inhibitors of viral maturation than

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integration. Treatment of virus-producing cells with ALLINIs leads to the generation of virions

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that have aberrant morphologies and substantially reduced infectivity27, 32, 33. Resistance

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mutations confirm that both the anti-integration and viral maturation effects are caused by

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ALLINIs binding to the IN LEDGF-pocket26, 33. These ALLINI-induced defects in viral

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maturation recall similar effects caused by some “Class II” IN mutations, which were defined as

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having a greater effect on viral maturation and/or reverse transcription than proviral

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integration34. Subsequent work has shown that some of these Class II IN mutants produce

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malformed capsids with similar morphologies to WT virions produced in the presence of

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ALLINIs32. 3 ACS Paragon Plus Environment


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The mechanism of this pharmacological block in viral maturation is most likely due to

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ALLINI-induced oligomerization of IN inside the developing virion, leading to a corruption of

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the capsid architecture rendering the virion non-infectious27-29, 32, 33, 35. If HIV-1 maturation is

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indeed sensitive to agents that can oligomerize IN, it is reasonable to speculate that other

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allosteric compounds that can bind outside of the LEDGF-binding pocket and trigger IN

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oligomerization could also have this “late” antiviral mechanism. In this study, we report the

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discovery and characterization of a new class of allosteric IN inhibitors, the t-butylsulfonamides,

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which share many of the biochemical and antiviral characteristics of known ALLINIs. However,

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experiments with virus and proteins harboring ALLINI-resistance mutants and binding studies

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with recombinant proteins strongly suggest that the t-butylsulfonamides make unique contacts

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with IN relative to known ALLINIs, and engage with residues that are outside of the LEDGF-

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binding pocket.

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RESULTS AND DISCUSSION:

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An HTS to identify new structural classes of ALLINIs was run using an IN·LEDGF

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displacement assay (Figure 1A). Hits were triaged through two orthogonal biochemical assays,

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ALLINI-induced multimerization and strand-transfer assays (Figures 1B and C), that take

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advantage of the multimodal effects of ALLINIs on IN structure and function25, 26. One of the

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more compelling hit series identified was a class of t-butylsulfonamides, exemplified by 1

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(Figure 2A). While structurally distinct from t-butoxyacid ALLINIs, such as 2 and 3 (Figure

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2A), 1 behaved similarly to known ALLINIs in a battery of biochemical assays: it blocked the

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interaction between IN and LEDGF (Figure 2B), inhibited LEDGF-independent IN-catalyzed

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DNA strand transfer (Figure 2C) and appeared to trigger a change in IN conformation and/or


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oligomeric state of two differently tagged full-length proteins that can be detected by a saturable

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increase in TR-FRET (Figures 2D).

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A characteristic feature of the antiviral mechanism of ALLINIs is that they are far more

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potent inhibitors of viral maturation than integration31-33, 36, 37. To determine whether 1 shared

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this antiviral mechanism, we compared its potency in a multi-cycle antiviral assay (which would

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be sensitive to inhibition at any step of the viral lifecycle) to its potency in a single-cycle assay

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(which is only sensitive to inhibition of the early steps of the viral lifecycle - entry through

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transcription). As expected, the t-butoxyacid ALLINI 2 is substantially more potent in the multi-

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cycle than the single-cycle assay (Figure 3A). Interestingly, 1 is also more potent in a multi-

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cycle assay (Figure 3B), suggesting that it too interferes with a post-integration step in the HIV

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lifecycle.

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To assess the antiviral properties of these compounds on later stages of the viral lifecycle

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(i.e. post-transcription), supernatant from infected cells used in the single-cycle assay (with and

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without compound treatment) was used to infect naïve cells in the absence of additional

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compound treatment. In cases where an antiretroviral agent inhibits a step in the viral lifecycle

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up through viral transcription, such as an NNRTI or INSTI, the early and late stage potencies

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should agree, since the early antiviral effect will have both prevented activation of the reporter

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gene in the single-cycle assay, as well as inhibited the production of new infectious virions

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(Figures S1A and B). In contrast, an antiretroviral agent that blocks a post-transcription step,

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such as an HIV protease inhibitor, will be ineffective in the single-cycle assay, but its efficacy

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will be revealed in the late stage assay (Figures S1A and B). As seen in Figures 3A and B, both

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1 and 2 are more potent in preventing the production of infectious virions at some post-

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transcription step than blocking HIV proviral integration, and this late stage effect corresponds 5 ACS Paragon Plus Environment


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with the multi-cycle antiviral potencies of both compounds. Thus, 1 shares the late antiviral

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effects of the structurally distinct t-butoxyacids, which is consistent with the t-butylsulfonamide

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acting as an ALLINI.

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Despite similarities in the biochemical and antiretroviral behavior between 1 and t-

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butoxyacid ALLINIs, important differences emerged between the two series that were

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inconsistent with the t-butylsulfonamides interacting with HIV integrase in a similar manner as

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the t-butoxyacids. We were unable to obtain a crystal structure of 1 bound to HIV IN CCD using

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either co-crystallization or soaking methods, including under conditions that are amenable to t-

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butoxyacids14, 33. In addition, computational molecular docking efforts were unable to provide a

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binding mode for the t-butylsulfonamides in the site shared by LEDGF and the t-butoxyacids that

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could adequately explain the SAR observed for this series (data not shown). To further

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characterize the interaction of 1 with IN, the effects of two t-butoxyacid-resistance mutations on

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its antiretroviral and biochemical integrase strand-transfer potencies were measured. Virus

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harboring the IN A128T mutation have been reported to provide modest resistance to ALLINIs,

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likely through attenuating the effects of IN oligomerization, rather than by weakening the affinity

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of t-butoxyacid binding36. In contrast, the IN T174I virus removes a key interaction between a

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protein hydrogen bond donor and the t-butoxyacid and replaces it with an unfavorable

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hydrophobic interaction, resulting in decreased compound affinity and dramatically reduced

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antiretroviral potency26, 33. Using the t-butoxyacid ALLINI 2 to benchmark these IN mutant viral

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strains, we demonstrated that the A128T and T174I mutations provide approximately nine-fold

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and 500-fold resistance to 2 in a multi-cycle antiviral assay (Figure 4A). Unexpectedly, while

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the A128T virus is still modestly resistant (~3-fold) to the antiviral effects of 1, virus harboring


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the T174I IN mutation is approximately five-fold more susceptible to the antiviral effects of the

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t-butylsulfonamide (Figure 4B).

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A similar pattern of resistance and susceptibility is observed using recombinant IN

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proteins in a biochemical strand-transfer assay (Figures 4C and D), where 1 is 4.6-fold less

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potent against A128T IN and 3.8-fold more potent against T174I IN. Hence, a mutation that is

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implicated in attenuating ALLINI-induced IN oligomerization provides modest resistance

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against 1 in biochemical and antiviral assays, and another resistance mutation that ablates a key

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interaction with the t-butoxyacid ALLINIs does not confer resistance to the t-butylsulfonamide

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1, suggesting that T174 does not contribute positively to its binding.

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Taking the inability to co-crystallize or model 1 into the IN CCD together with the lack

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of resistance provided by the T174I mutation, we questioned whether the t-butylsulfonamide

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ALLINIs engage with IN in the same manner as the t-butoxyacids. To better understand the

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binding behavior of this series, we took advantage of the broad tolerance of the t-

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butylsulfonamides to substitutions off the propionyl moiety (will be the subject of a separate

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publication) to synthesize 4, a fluorescence polarization (FP) probe of IN (FLIPPIN; Figures 5A

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and B). Titration of WT full-length IN in the presence of the FLIPPIN probe 4 shows a saturable

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increase in FP that is completely blocked by addition of excess 1, suggesting that the interaction

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of 4 with IN is specific (Figure 5C). Fitting these FP data using Equation 1 (Materials and

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Methods) yields an apparent KD value of 264 ± 19 nM. The Hill coefficient of binding is > 1

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(1.8 ± 0.2), suggesting that at least two molecules of IN bind to each molecule of 4, which is

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consistent with the ability of the t-butylsulfonamides to trigger IN multimerization (Figure 2D).

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In contrast to full-length IN, the IN CCD shows no evidence of binding 4, even at protein

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concentrations as high as 5 µM (Figure 5C). The absence of high affinity binding of 1 to the IN 7 ACS Paragon Plus Environment


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CCD was confirmed using surface plasmon resonance (Figure S2A). In contrast, the t-

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butoxyacid 2 binds this CCD construct with high affinity (Figure S2B). Thus, unlike the t-

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butoxyacid ALLINIs, the IN CCD is not sufficient to bind the t-butylsulfonamides, indicating

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that IN residues outside the catalytic core domain make important contacts with this series and/or

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are needed for IN to adopt a conformation that is competent to bind this class of ALLINIs.

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Taking advantage of the ability to continuously monitor fluorescence polarization, the

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kinetics of 4 binding to full-length IN were measured (Figures S3A and B). The observed first-

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order binding constant (kobs = 3.1 x 10-4 ± 0.07 x 10-4 s-1), dissociation rate constant (koff = 1.8 x

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10-4 ± 0.17 x 10-4 s-1) and calculated second-order association rate constant (kon = 554 M-1s-1) are

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all relatively slow, suggesting that there is a significant kinetic barrier in one or more of the

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binding steps of 4 to IN. Thus, despite its modest binding affinity, 4 has a relatively long

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residence time (1/ koff = 92 min) when bound to IN, a property that has been correlated with

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efficacious pharmaceutical agents37.

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To further characterize the t-butylsulfonamide binding site, the ability of LEDGF and the

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two classes of ALLINIs to displace the probe molecule 4 from full-length IN was measured.

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Consistent with t-butylsulfonamides as inhibitors of the IN·LEDGF interaction, LEDGF can

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block the formation of the IN·4 complex (Figure 6A), and the t-butylsulfonamide 1 can displace

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the homologous FP probe in a dose-dependent manner from WT full-length IN (Figure 6B). In

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addition, the t-butoxyacid ALLINIs 2 (Figure S4) and 3 (Figure 6B) were also able to displace

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the FP probe from IN. Thus, despite differences in their binding modes, the t-butylsulfonamides,

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LEDGF and the t-butoxyacids bind mutually exclusively to IN. To determine if the

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displacement of the FP probe by the t-butoxyacids was through specific interactions with IN, an

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FP assay using T174I full-length IN was developed (Figure S5). Consistent with the ability of 8 ACS Paragon Plus Environment

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the t-butylsulfonamide 1 to inhibit strand-transfer reactions catalyzed by IN and inhibit

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replication of virus harboring that mutation, the FP probe 4 specifically binds T174I IN. Like

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WT IN, 1 can displace 4 from T174I IN in a dose-dependent manner (Figure 6C). However, the

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introduction of the T174I mutation weakens the potencies of 2 and 3 to displace the FP probe

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(Figures S4 and 6C). Thus, the displacement of the t-butylsulfonamide probe by the t-

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butoxyacid ALLINIs is due to specific interactions between the t-butoxyacid and full-length IN,

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and not to non-specific effects of the compound on the protein or probe.

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Despite sharing many of the biochemical and antiviral properties of ALLINIs, several lines of evidence suggest that the t-butylsulfonamide series does not share the same binding

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determinants on IN as the t-butoxyacids. Recombinant IN or virus harboring a T174I mutation

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are not resistant to the effects of the t-butylsulfonamides. In addition, a t-butylsulfonamide FP

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probe that specifically binds to full-length IN was incapable of binding to the IN CCD. Hence,

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unlike the t-butoxyacids, which bind to the IN CCD with high affinity14, 33, the t-

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butylsulfonamides likely make important contacts with residues in either the N- or C-terminal

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domains. From these data, one cannot distinguish if the putative t-butylsulfonamide pocket is

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separate from the LEDGF-binding site, triggering allosteric changes that prevent LEDGF and t-

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butoxyacids from binding IN, or whether the t-butylsulfonamides partially occupy the LEDGF

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pocket, but requires other contacts outside of the CCD for tight binding. Other IN pockets that

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are capable of binding small molecules have been identified using fragment screening38, 39, and a

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pocket along the CCD dimer interface that binds sucrose was revealed by x-ray

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crystallography40, 41. The natural product kuwanon-L was identified as a putative ligand to this

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sucrose-binding pocket by molecular docking studies, and possesses ALLINI-like properties in

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biochemical assays with double-digit micromolar potencies, including inhibiting the LEDGF·IN 9 ACS Paragon Plus Environment


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interaction42. However, because of the demonstrated insufficiency of the CCD to support high-

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affinity binding of the t-butylsulfonamides, or to co-crystallize this series with the CCD under

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conditions that revealed the fragment- and sucrose binding-pockets, it is difficult in the absence

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of other data to verify or exclude binding of 1 to these other CCD pockets. In addition, the

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conformational and oligomeric plasticity of IN9, 31 suggest that other compound binding sites

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could be induced. Recent structural data have revealed interactions between t-butoxyacids and

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both CCD and C-terminal domain43, which offers a structural explanation for compound-induced

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multimerization of HIV IN and raises the possibility that the t-butylsulfonamides require

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interactions with this region of the C-terminal domain for binding. In lieu of a curative regimen, there will be a continuing need to develop new

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antiretroviral agents to treat patients living with lifelong, persistent HIV infections. The

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characterization of the t-butoxyacid ALLINI mode of action revealed a previously unexploited

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vulnerability of HIV assembly to compound-induced IN multimerization. Despite using a

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screening strategy based on the original anti-LEDGF hypothesis, the identification of the t-

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butylsulfonamide ALLINIs demonstrate that additional chemotypes that affect late steps in the

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viral lifecycle and bearing different resistance profiles from other ALLINIs can be discovered.

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Future efforts that specifically screen for compounds that can induce IN or GagPol

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multimerization are likely to reveal new chemotypes that can disrupt viral assembly, providing

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additional pharmacological tools to dissect these later phases of the HIV lifecycle, as well as

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starting points for medicinal chemistry efforts to develop the next generation of antiretroviral

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drugs.

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MATERIALS AND METHODS:


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Recombinant Proteins. E. coli expression and purification of untagged, full-length IN44,

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N-terminal His-tagged (NTH) and C-terminal FLAG-tagged full-length IN, NTH-CCD IN and

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C-terminal FLAG-tagged LEDGF45 have been described.

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Synthesis of Chemical Probes. The preparation of 2 has been described26. The synthesis

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of t-butylsulfonamide 1, t-butoxyacid 3 and the FLIPPIN probe 4 are described in Supplemental

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Methods.

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Integrase Strand-Transfer Assay. The IN strand-transfer assay was run as previously

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described; compounds were added to untagged full-length IN prior to assembly onto the viral

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DNA oligonucleotide12.

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Assessing antiviral potency in a multi-cycle HIV-1 infection assay. HIV-1 replication

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was monitored using MT4-gag-GFP clone D3 (hereafter designate MT4-GFP), which are MT-4

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cells modified to express an HIV tat- and rev- dependent GFP reporter gene. Productive infection

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of MT4-GFP cells with HIV-1 results in GFP expression approximately 24 h post-infection.

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MT4-GFP cells were maintained at 37ºC, 5% CO2 and 90% relative humidity in RPMI

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1640 supplemented with 10% fetal bovine serum, 100 U mL-1 penicillin/streptomycin, and 400

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µg mL-1 G418 to maintain the reporter gene. For infections, MT4-GFP cells were placed in the

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same medium lacking G418 and infected overnight with H9/IIIB virus at an approximate

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multiplicity of infection of 0.01 in the same incubation conditions. Cells were washed and re-

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suspended in either RPMI 1640 containing 0.1% or 10% fetal bovine serum at 1.6 x 105 cells

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mL-1. Compound plates were prepared by dispensing compounds dissolved in DMSO into wells

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of 384 well poly-D-lysine-coated plates (0.2 µl/well) using an ECHO acoustic dispenser

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(Labcyte Inc.). Each compound was tested in a 10-point serial 3-fold dilution (typical final



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concentrations: 8.4 µM – 0.43 nM). Controls included no inhibitor (DMSO only) and a

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combination of three antiviral agents (efavirenz, indinavir, and an integrase strand transfer

3

inhibitor at final concentrations of 4 µM each). Cells were added (50 µL/well) to compound

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plates and the infected cells were maintained at 37ºC, 5% CO2 and 90% relative humidity.

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5

Infected cells were quantified at two time points, ~48 h and ~72 h post-infection, by

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counting the green cells in each well using an Acumen eX3 scanner (TTP Labtech). The

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increase in the number of green cells over ~24 h period gives the reproductive ratio, R0, which is

8

typically 5-15 and has been shown experimentally to be in logarithmic phase (data not

9

shown). Inhibition of R0 was calculated for each well, and IC50 values were determined by non-

10

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linear four-parameter curve fitting. Fluorescence Polarization Probe of Integrase (FLIPPIN). To determine the apparent

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affinity of an HIV integrase construct for the FLIPPIN probe (4), DMSO or saturating amounts

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of unlabeled competitor ALLINI 1 (at a final concentration of 100 µM) in DMSO was

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transferred to a black 384-well Proxiplate (PerkinElmer) using an ECHO dispenser. A solution

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of the FLIPPIN probe in buffer (27.8 mM HEPES, pH 7.3, 27.8 mM MgCl2, 57.1 mM NaCl, 5.6

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mM β-mercaptoethanol, and 0.1 mg mL-1 acetylated BSA) was added to each well, followed by a

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solution of an HIV IN construct in assay buffer. The final binding reactions contained 4 nM

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FLIPPIN probe, 0 – 5 µM HIV IN protein construct and 2% DMSO. Binding reactions were

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incubated at ambient temperature for 3 h with gentle shaking, and fluorescence polarization (FP)

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of the fluorescein-labeled probe was measured on an Envision plate reader (PerkinElmer).

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Apparent binding affinities (KDapp) were calculated by plotting FP vs. protein concentration (in

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both the presence and absence of saturating unlabeled competitor ALLINI) in GraphPad Prism,

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fitting these data to Equation 1: 12 ACS Paragon Plus Environment

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1


=

( )

( [])∗"

+ $%&'

(1)

2

Where FP, FPbkgd and FPmax are the FP of the probe at a given [IN], in the absence of IN, and

3

with saturating IN, respectively, KDapp is the [IN] that yields a half-maximal change in the FP

4

signal and h is the Hill coefficient.

5

To determine the apparent potencies of ALLINIs or LEDGF to displace the FLIPPIN

6

probe, the same protocol was used, except the final concentration of each HIV integrase protein

7

construct was fixed at either 1 µM (competition with ALLINIs) or 2.5 µM (competition with

8

LEDGF). Compound IC50 values were calculated by plotting FP vs. log[compound] in GraphPad

9

Prism and fitting the resulting dose-response curves to Equation S1.



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1

Associated Content

2

Supporting Information

3

The chemical syntheses of 1, 3 and 4, methods for IN-LEDGF interaction and IN

4

multimerization assays, measurement of FLIPPIN probe binding kinetics, assessment of single-

5

cycle antiviral potencies and measurement of ALLINI binding to IN CCD by surface plasmon

6

resonance (SPR), validation of early and late antiviral assays, SPR data, FLIPPIN probe binding

7

kinetics, displacement of FLIPPIN probe by 2 and FLIPPIN assays with T174I IN.

8 9

Author Information

10

Corresponding Author

11

*Current address: Molecular & Cellular Pharmacology, Lead Discovery, Janssen

12

Research & Development, Spring House, PA, 19477, USA.

13

E-mail: [email protected]

14

Notes: The authors do not have any conflicts of interest to disclose.

15

Funding Sources: This work was supported by Merck & Co., Inc.

16


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1


Figure 1: Biochemical Assays Used to Characterize ALLINI Compounds. A.

2

IN·LEDGF-FLAG interaction assay. B. ALLINI-induced multimerization assay. C. IN strand-

3

transfer assay.

4



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1

Figure 2: Comparison of the Putative ALLINI t-Butylsulfonamide 1 with t-

2

Butoxyacid ALLINIs in a Panel of Biochemical Assays. A. Structures of the t-

3

butylsulfonamide ALLINI HTS hit 1 and the t-butyoxyacid ALLINIs 2 and 3. B. Displacement

4

of C-FLAG LEDGF from IN. 1 (blue circles): IC50 = 1.5 ± 0.1 µM; 2 (green triangles): IC50 =

5

0.036 ± 0.002 µM. C. Inhibition of LEDGF-independent IN strand-transfer activity. 1: IC50 =

6

0.33 ± 0.04 µM; 2: IC50 = 0.028 ± 0.002 µM. D. Dose-dependent multimerization of NTH- and

7

C-FLAG-IN by ALLINI compounds. 1: EC50 = 0.45 ± 0.06 µM, maximum TR-FRET increase

8

from baseline = 240 ± 4%; 2: EC50 = 0.088 ± 0.01 µM, maximum TR-FRET increase from

9

baseline = 550 ± 5%; 3 (gray squares): EC50 = 2.6 ± 0.18 µM, maximum TR-FRET increase

10

from baseline = 564 ± 9%.

11


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1


Figure 3: Single and Multi-cycle Antiviral Activity of ALLINIs. The potencies of 1

2

and 2 were assessed in a single-cycle “early” antiviral assay (blue triangles) that is sensitive to

3

inhibitors of the viral lifecycle up to and including HIV transcription. Supernatant from these

4

single-cycle assays was then used to infect a fresh set of cells to assess the relative number of

5

infectious particles produced under each treatment condition (“late” antiviral assay; red squares).

6

Both ALLINIs were also assessed in a multi-cycle antiviral assay (green circles) that is sensitive

7

to inhibitors of any stage of the viral lifecycle. These assays were conducted in the presence of

8

10% FBS. A. 2: early antiviral IC50 = 1.5 ± 0.1 µM; late antiviral IC50 = 0.035 ± 0.003 µM;

9

multi-cycle IC50 = 0.039 ± 0.005 µM. B. 1: early antiviral IC50 = 42 ± 5.2 µM; late antiviral IC50

10

= 7.0 ± 1.2 µM; multi-cycle IC50 = 8.6 ± 1.5 µM.

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 4: Differentiating the Two Structural Classes of ALLINI Compounds by

2

Their Biochemical and Antiviral Mutant Profiles. 2 (A.) and 1 (B.) were tested in dose-

3

response titrations in multi-cycle antiviral assays in the presence of 0.1% FBS against WT (blue

4

circles), A128T (green triangles) and T174I (red squares) HIV strains. 2: WT IC50 = 0.010 ±

5

0.001 µM; A128T IC50 = 0.093 ± 0.015 µM; T174I IC50 = 5.4 ± 1.0 µM. 1: WT IC50 = 1.3 ± 0.2

6

µM; A128T IC50 = 4.1 ± 0.9 µM; T174I IC50 = 0.26 ± 0.06 µM. Inhibition of WT (blue circles),

7

A128T (green triangles) and T174I (red squares) NTH-IN enzymatic strand-transfer activity by 2

8

(C.) and 1 (D.) using IN strand-transfer assay were assessed in dose-response titrations. 2: WT

9

IC50 = 31.6 ± 4.0 nM; A128T IC50 = 39.7 ± 3.8 nM; T174I IC50 = 758 ± 43 nM. 1: WT IC50 =

10

995 ± 210 nM; A128T IC50 = 4529 ± 1354 nM; T174I IC50 = 251 ± 46 nM.


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Figure 5: Fluorescence Polarization Probe of IN (FLIPPIN) Assay. A. Assay

2

schematic. B. Structure of the FLIPPIN probe (4). C. The FLIPPIN probe (4 nM) was titrated

3

with full-length WT IN (blue circles), and the resulting data fit to Equation 1 (see Materials and

4

Methods): EC50 = 264 ± 19 nM, FPmax = 324 ± 5 mP, FPbkgd = 77 ± 8 mP and h = 1.8 ± 0.2.

5

Addition of 100 µM 1 to the binding reaction abolished binding of 4 to full-length WT IN (green

6

triangles): EC50 > 5000 nM. Titration of the HIV integrase core catalytic domain (IN CCD) in

7

the absence of competitor compound did not result in an increase in FP of the FLIPPIN probe

8

over background (red squares): EC50 > 5000 nM.

9



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1

Figure 6: Dose-dependent Displacement of FLIPPIN Probe from WT and T174I IN.

2

A. Displacement of FLIPPIN probe 4 from 2.5 µM WT NTH-IN by LEDGF-FLAG: IC50 = 1400

3

± 150 nM. A t-butylsulfonamide (1, blue circles) and a t-butoxyacid (3, green triangles) were

4

titrated in the FLIPPIN assay using WT (B.) and T174I (C.) 1 µM NTH-IN (see Materials and

5

Methods). WT potencies: 1 IC50 = 412 ± 44.5 nM, 3 IC50 = 1000 ± 78 nM; T174I potencies: 1

6

IC50 = 733 ± 40.3 nM, 3 IC50 > 100,000 nM.

7


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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

2


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1. UNAIDS. (2016) Global AIDS Update 2016. 2. Chiasson, M. A., Berenson, L., Li, W., Schwartz, S., Singh, T., Forlenza, S., Mojica, B. A., and Hamburg, M. A. (1999) Declining HIV/AIDS mortality in New York City. JAIDS 21, 59-64. 3. Hammer , S. M., Katzenstein , D. A., Hughes , M. D., Gundacker , H., Schooley , R. T., Haubrich , R. H., Henry , W. K., Lederman , M. M., Phair , J. P., Niu , M., Hirsch , M. S., and Merigan , T. C. (1996) A trial comparing nucleoside monotherapy with combination therapy in HIV-infected adults with CD4 cell counts from 200 to 500 per cubic millimeter. New Engl. J. Med. 335, 10811090. 4. Hammer , S. M., Squires , K. E., Hughes , M. D., Grimes , J. M., Demeter , L. M., Currier , J. S., Eron , J. J. J., Feinberg , J. E., Balfour , H. H. J., Deyton , L. R., Chodakewitz , J. A., Fischl , M. A., Phair , J. P., Pedneault , L., Nguyen , B.-Y., and Cook , J. C. (1997) A controlled trial of two nucleoside analogues plus indinavir in persons with Human Immunodeficiency Virus infection and CD4 cell counts of 200 per cubic millimeter or less. New Engl. J. Med. 337, 725-733. 5. Cohen , M. S., Chen , Y. Q., McCauley , M., Gamble , T., Hosseinipour , M. C., Kumarasamy , N., Hakim , J. G., Kumwenda , J., Grinsztejn , B., Pilotto , J. H. S., Godbole , S. V., Mehendale , S., Chariyalertsak , S., Santos , B. R., Mayer , K. H., Hoffman , I. F., Eshleman , S. H., PiwowarManning , E., Wang , L., Makhema , J., Mills , L. A., de Bruyn , G., Sanne , I., Eron , J., Gallant , J., Havlir , D., Swindells , S., Ribaudo , H., Elharrar , V., Burns , D., Taha , T. E., Nielsen-Saines , K., Celentano , D., Essex , M., and Fleming , T. R. (2011) Prevention of HIV-1 infection with early antiretroviral therapy. New Engl. J. Med. 365, 493-505. 6. Centers for Disease Control & Prevention (2015) HIV Surveillance Report - Diagnosis of HIV Infection in the United States and Dependent Areas, 2014. 7. Siliciano, J. D., Kajdas, J., Finzi, D., Quinn, T. C., Chadwick, K., Margolick, J. B., Kovacs, C., Gange, S. J., and Siliciano, R. F. (2003) Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat. Med. 9, 727-728. 8. Collins, S., Grant, P., and Shafer, R. (2015) Modifying antiretroviral therapy in virologically suppressed HIV-1-infected patients. Drugs, 1-24. 9. Lesbats, P., Engelman, A. N., and Cherepanov, P. (2016) Retroviral DNA integration. Chem. Rev.116, 12730-12757. 10. Summa, V., Petrocchi, A., Bonelli, F., Crescenzi, B., Donghi, M., Ferrara, M., Fiore, F., Gardelli, C., Gonzalez Paz, O., Hazuda, D. J., Jones, P., Kinzel, O., Laufer, R., Monteagudo, E., Muraglia, E., Nizi, E., Orvieto, F., Pace, P., Pescatore, G., Scarpelli, R., Stillmock, K., Witmer, M. V., and Rowley, M. (2008) Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV-AIDS infection. J. Med. Chem. 51, 5843-5855. 11. Espeseth, A. S., Felock, P., Wolfe, A., Witmer, M., Grobler, J., Anthony, N., Egbertson, M., Melamed, J. Y., Young, S., Hamill, T., Cole, J. L., and Hazuda, D. J. (2000) HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase. Proc. Natl. Acad. Sci U.S.A. 97, 11244-11249. 12. Grobler, J. A., Stillmock, K., Hu, B., Witmer, M., Felock, P., Espeseth, A. S., Wolfe, A., Egbertson, M., Bourgeois, M., Melamed, J., Wai, J. S., Young, S., Vacca, J., and Hazuda, D. J. (2002) Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes. Proc. Natl. Acad. Sci U.S.A. 99, 6661-6666. 13. Hare, S., Gupta, S. S., Valkov, E., Engelman, A., and Cherepanov, P. (2010) Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232-236. 14. Christ, F., Voet, A., Marchand, A., Nicolet, S., Desimmie, B. A., Marchand, D., Bardiot, D., Van der Veken, N. J., Van Remoortel, B., Strelkov, S. V., De Maeyer, M., Chaltin, P., and Debyser, Z. (2010) Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication. Nat. Chem. Biol. 6, 442-448. 15. Ge, H., Si, Y., and Roeder, R. G. (1998) Isolation of cDNAs encoding novel transcription coactivators p52 and p75 reveals an alternate regulatory mechanism of transcriptional activation. EMBO J. 17, 6723-6729. 27 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

16. De Rijck, J., Bartholomeeusen, K., Ceulemans, H., Debyser, Z., and Gijsbers, R. (2010) Highresolution profiling of the LEDGF/p75 chromatin interaction in the ENCODE region. Nucleic Acids Res. 38, 6135-6147. 17. Turlure, F., Maertens, G., Rahman, S., Cherepanov, P., and Engelman, A. (2006) A tripartite DNAbinding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin in vivo. Nucleic Acids Res. 34, 1653-1665. 18. Cherepanov, P., Maertens, G., Proost, P., Devreese, B., Van Beeumen, J., Engelborghs, Y., De Clercq, E., and Debyser, Z. (2003) HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells. J. Biol. Chem. 278, 372-381. 19. Maertens, G., Cherepanov, P., Pluymers, W., Busschots, K., De Clercq, E., Debyser, Z., and Engelborghs, Y. (2003) LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells. J. Biol. Chem. 278, 33528-33539. 20. Cherepanov, P., Devroe, E., Silver, P. A., and Engelman, A. (2004) Identification of an evolutionarily conserved domain in human lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF/p75) that binds HIV-1 integrase. J. Biol. Chem. 279, 48883-48892. 21. Ciuffi, A., Llano, M., Poeschla, E., Hoffmann, C., Leipzig, J., Shinn, P., Ecker, J. R., and Bushman, F. (2005) A role for LEDGF/p75 in targeting HIV DNA integration. Nat. Med. 11, 1287-1289. 22. Llano, M., Saenz, D. T., Meehan, A., Wongthida, P., Peretz, M., Walker, W. H., Teo, W., and Poeschla, E. M. (2006) An essential role for LEDGF/p75 in HIV integration. Science 314, 461464. 23. Fader, L. D., Malenfant, E., Parisien, M., Carson, R., Bilodeau, F., Landry, S., Pesant, M., Brochu, C., Morin, S., Chabot, C., Halmos, T., Bousquet, Y., Bailey, M. D., Kawai, S. H., Coulombe, R., LaPlante, S., Jakalian, A., Bhardwaj, P. K., Wernic, D., Schroeder, P., Amad, M., Edwards, P., Garneau, M., Duan, J., Cordingley, M., Bethell, R., Mason, S. W., Bos, M., Bonneau, P., Poupart, M. A., Faucher, A. M., Simoneau, B., Fenwick, C., Yoakim, C., and Tsantrizos, Y. (2014) Discovery of BI 224436, a noncatalytic site integrase inhibitor (NCINI) of HIV-1. ACS Med. Chem. Lett. 5, 422-427. 24. Fenwick, C., Amad, M. a., Bailey, M. D., Bethell, R., Bös, M., Bonneau, P., Cordingley, M., Coulombe, R., Duan, J., Edwards, P., Fader, L. D., Faucher, A.-M., Garneau, M., Jakalian, A., Kawai, S., Lamorte, L., LaPlante, S., Luo, L., Mason, S., Poupart, M.-A., Rioux, N., Schroeder, P., Simoneau, B., Tremblay, S., Tsantrizos, Y., Witvrouw, M., and Yoakim, C. (2014) Preclinical profile of BI 224436, a novel HIV-1 non-catalytic-site integrase inhibitor. Antimicrob. Agents Chemother. 58, 3233-3244. 25. Kessl, J. J., Jena, N., Koh, Y., Taskent-Sezgin, H., Slaughter, A., Feng, L., de Silva, S., Wu, L., Le Grice, S. F. J., Engelman, A., Fuchs, J. R., and Kvaratskhelia, M. (2012) Multimode, cooperative mechanism of action of allosteric HIV-1 integrase inhibitors. J. Biol. Chem. 287, 16801-16811. 26. Tsiang, M., Jones, G. S., Niedziela-Majka, A., Kan, E., Lansdon, E. B., Huang, W., Hung, M., Samuel, D., Novikov, N., Xu, Y., Mitchell, M., Guo, H., Babaoglu, K., Liu, X., Geleziunas, R., and Sakowicz, R. (2012) New class of HIV-1 integrase (IN) inhibitors with a dual mode of action. J. Biol. Chem. 287, 21189-21203. 27. Desimmie, B., Schrijvers, R., Demeulemeester, J., Borrenberghs, D., Weydert, C., Thys, W., Vets, S., Van Remoortel, B., Hofkens, J., De Rijck, J., Hendrix, J., Bannert, N., Gijsbers, R., Christ, F., and Debyser, Z. (2013) LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions. Retrovirology 10, 57. 28. Gupta, K., Brady, T., Dyer, B. M., Malani, N., Hwang, Y., Male, F., Nolte, R. T., Wang, L., Velthuisen, E., Jeffrey, J., Van Duyne, G. D., and Bushman, F. D. (2014) Allosteric inhibition of Human Immunodeficiency Virus integrase: Late block during replication and abnormal multimerization involving specific protein domains. J. Biol. Chem. 289, 20477-20488. 29. Feng, L., Dharmarajan, V., Serrao, E., Hoyte, A., Larue, R. C., Slaughter, A., Sharma, A., Plumb, M. R., Kessl, J. J., Fuchs, J. R., Bushman, F. D., Engelman, A. N., Griffin, P. R., and Kvaratskhelia, M. (2016) The competitive interplay between allosteric HIV-1 integrase inhibitor BI/D and 28 ACS Paragon Plus Environment

Page 28 of 31

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50


LEDGF/p75 during the early stage of HIV-1 replication adversely affects inhibitor potency. ACS Chem. Biol. 11, 1313-1321. 30. Deng, N., Hoyte, A., Mansour, Y. E., Mohamed, M. S., Fuchs, J. R., Engelman, A. N., Kvaratskhelia, M., and Levy, R. (2016) Allosteric HIV-1 integrase inhibitors promote aberrant protein multimerization by directly mediating inter-subunit interactions: Structural and thermodynamic modeling studies. Protein Sci. 25, 1911-1917. 31. Hayouka, Z., Rosenbluh, J., Levin, A., Loya, S., Lebendiker, M., Veprintsev, D., Kotler, M., Hizi, A., Loyter, A., and Friedler, A. (2007) Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium. Proc. Natl. Acad. Sci U.S.A. 104, 8316-8321. 32. Jurado, K. A., Wang, H., Slaughter, A., Feng, L., Kessl, J. J., Koh, Y., Wang, W., Ballandras-Colas, A., Patel, P. A., Fuchs, J. R., Kvaratskhelia, M., and Engelman, A. (2013) Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation. Proc. Natl. Acad. Sci U.S.A. 110, 8690-8695. 33. Le Rouzic, E., Bonnard, D., Chasset, S., Bruneau, J.-M., Chevreuil, F., Le Strat, F., Nguyen, J., Beauvoir, R., Amadori, C., Brias, J., Vomscheid, S., Eiler, S., Levy, N., Delelis, O., Deprez, E., Saib, A., Zamborlini, A., Emiliani, S., Ruff, M., Ledoussal, B., Moreau, F., and Benarous, R. (2013) Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage. Retrovirology 10, 144. 34. Engelman, A. (1999) In vivo analysis of retroviral integrase structure and function. In Advances in Virus Research (Karl Rlaramorosch, F. A. M., and Aaron, J. S., Eds.), pp 411-426, Academic Press. 35. Kessl, J. J., Kutluay, S. B., Townsend, D., Rebensburg, S., Slaughter, A., Larue, R. C., Shkriabai, N., Bakouche, N., Fuchs, J. R., Bieniasz, P. D., and Kvaratskhelia, M. (2016) HIV-1 integrase binds the viral RNA genome and is essential during virion morphogenesis. Cell 166, 1257-1268 e1212. 36. Feng, L., Sharma, A., Slaughter, A., Jena, N., Koh, Y., Shkriabai, N., Larue, R. C., Patel, P. A., Mitsuya, H., Kessl, J. J., Engelman, A., Fuchs, J. R., and Kvaratskhelia, M. (2013) The A128T resistance mutation reveals aberrant protein multimerization as the primary mechanism of action of allosteric HIV-1 integrase inhibitors. J. Biol. Chem. 288, 15813-15820. 37. Tummino, P. J., and Copeland, R. A. (2008) Residence time of receptor−ligand complexes and its effect on biological function. Biochemistry 47, 5481-5492. 38. Rhodes, D. I., Peat, T. S., Vandegraaff, N., Jeevarajah, D., Le, G., Jones, E. D., Smith, J. A., Coates, J. A., Winfield, L. J., Thienthong, N., Newman, J., Lucent, D., Ryan, J. H., Savage, G. P., Francis, C. L., and Deadman, J. J. (2011) Structural basis for a new mechanism of inhibition of HIV-1 integrase identified by fragment screening and structure-based design. Antiviral Chem. Chemother.21, 155-168. 39. Wielens, J., Headey, S. J., Deadman, J. J., Rhodes, D. I., Le, G. T., Parker, M. W., Chalmers, D. K., and Scanlon, M. J. (2011) Fragment-based design of ligands targeting a novel site on the integrase enzyme of Human Immunodeficiency Virus-1. ChemMedChem 6, 258-261. 40. Wielens, J., Headey, S. J., Jeevarajah, D., Rhodes, D. I., Deadman, J., Chalmers, D. K., Scanlon, M. J., and Parker, M. W. (2010) Crystal structure of the HIV-1 integrase core domain in complex with sucrose reveals details of an allosteric inhibitory binding site. FEBS Lett. 584, 1455-1462. 41. Tintori, C., Esposito, F., Morreale, F., Martini, R., Tramontano, E., and Botta, M. (2015) Investigation on the sucrose binding pocket of HIV-1 Integrase by molecular dynamics and synergy experiments. Bioorg. Med. Chem. Lett. 25, 3013-3016. 42. Esposito, F., Tintori, C., Martini, R., Christ, F., Debyser, Z., Ferrarese, R., Cabiddu, G., Corona, A., Ceresola, E. R., Calcaterra, A., Iovine, V., Botta, B., Clementi, M., Canducci, F., Botta, M., and Tramontano, E. (2015) Kuwanon-L as a new allosteric HIV-1 integrase inhibitor: Molecular modeling and biological evaluation. ChemBioChem 16, 2507-2512. 43. Gupta, K., Turkki, V., Sherrill-Mix, S., Hwang, Y., Eilers, G., Taylor, L., McDanal, C., Wang, P., Temelkoff, D., Nolte, R. T., Velthuisen, E., Jeffrey, J., Van Duyne, G. D., and Bushman, F. D.



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(2016) Structural basis for inhibitor-induced aggregation of HIV integrase. PLOS Biol. 14, e1002584. 44. Hazuda, D. J., Hastings, J. C., Wolfe, A. L., and Emini, E. A. (1994) A novel assay for the DNA strand-transfer reaction of HIV-1 integrase. Nucleic Acids Res. 22, 1121-1122. 45. Tsiang, M., Jones, G. S., Hung, M., Mukund, S., Han, B., Liu, X., Babaoglu, K., Lansdon, E., Chen, X., Todd, J., Cai, T., Pagratis, N., Sakowicz, R., and Geleziunas, R. (2009) Affinities between the binding partners of the HIV-1 integrase dimer-lens epithelium-derived growth factor (IN DimerLEDGF) complex. J. Biol. Chem.284, 33580-33599.

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