Discovery of a Distinct Chemical and Mechanistic Class of Allosteric

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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

4

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

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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

17

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

18

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

19

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

20

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

22

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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