WO2017040370A1

WO2017040370A1 - Small-molecule inhibitors of hiv-1 replication

Info

Publication number: WO2017040370A1
Application number: PCT/US2016/049199
Authority: WO
Inventors: Susana T. VALENTE; Suzie THENIN-HOUSSIER
Original assignee: Scripps Research Institute
Current assignee: Scripps Research Institute
Priority date: 2015-09-03
Filing date: 2016-08-29
Publication date: 2017-03-09
Anticipated expiration: 2018-03-03

Abstract

We developed a time-resolved fluorescence resonance energy transfer high-throughput screening assay (HTS-TR-FRET), to identify inhibitors of capsid dimerization, using the C-terminal domain (CTD) of HIV-1 capsid. This assay was used to screen the Library of Pharmacologically Active Compounds, composed of 1,280 in vivo active drugs, and identified Ebselen, an organoselenium compound, as a potent inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of Ebselen with the HIV-1 capsid CTD and dimer dissociation when Ebselen is in two-fold molar excess. Electrospray ionization mass spectrometry (ESI-MS) reveals Ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys 198 and Cys 218. This compound presents anti-HIV activity in single- and multiple-round of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells.

Description

SMALL-MOLECULE INHIBITORS OF HIV-1 REPLICATION

CROSS-REFERENCE TO RELATED APPLICATION

The application claims the priority of U.S. provisional application Ser. No. 62/213,771 , filed Sept. 3, 2015, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under 1R01 All 00685-01 awarded by the NIH-NIAID. The government has certain rights in the invention.

BACKGROUND

The HIV-1 mature conical core consists of 1 ,500 capsid (CA) monomers assembled into 250 hexamers and 12 pentamers. The capsid plays essential roles during the viral life cycle, e.g. the precise uncoating process of the capsid is tightly associated with reverse transcription [1 , 2], and capsid assembly and maturation are essential for viral particle integrity and infectivity [3, 4]. Antiretroviral therapy (ART) for HIV treatment consists of a "cocktail" or a combination of at least three drugs drawn from over 26 FDA-approved drugs, which fall in the categories of non- nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (Pis), integrase inhibitors (INIs), fusion inhibitors (FIs) and entry inhibitors. None of these drugs directly targets the assembly of the viral capsid protein, and given that the precise assembly of the HIV-1 capsid protein is crucial for HIV-1 replication, it represents an attractive target for drug development.

The viral capsid is synthetized from a 55 kDa Gag precursor, composed of three folded proteins [matrix (MA), capsid (CA) and nucleocapsid (NC)] and 3 small peptides [spacer peptides 1 and 2 (SP1 and SP2) and p6]. The Gag polyprotein plays an important role in membrane binding and in Gag-Gag lattice interaction in immature virions. Upon budding and release from the host cell, HIV maturation is initiated by proteolytic cleavage of Gag by the viral protease, resulting in the separation of CA from other functional domains of Gag, followed by the assembly of mature fullerene-like conical capsid. The viral capsid plays a critical role in both early and late events in the HIV-1 life cycle. Early on, fusion of the virus and target cell membranes triggers disassembly or uncoating of the conical capsid, which promotes completion of reverse transcription and synthesis of viral cDNA [1 , 2, 5]. Although the uncoating process is not completely understood, it seems that the HIV- 1 capsid's stability and integrity during the early stages of infection is essential for efficient reverse transcription and infectivity. Mutations in CA that affect its stability compromise HIV- 1 uncoating and infection [6-9]. Furthermore, two cellular restriction factors have been shown to inhibit HIV-1 infection by targeting the viral capsid during the uncoating process. Tripartite motif 5-alpha (TRIM5a) promotes a rapid and premature disassembly of viral capsids, thereby abrogating productive reverse transcription [10- 13], while the interferon-inducible MxB protein prevents uncoating by stabilizing the HIV- 1 core during infection, limiting integration of viral DNA [14-16]. During the late stages of the viral life cycle, capsid assembly and maturation have been shown to be essential for the formation of infectious viral particles, and mutagenesis studies have shown that mutations in capsid are detrimental for HIV-1 assembly and particle release [8, 17-23].

The CA protein consists of an independently folded N-terminal domain (NTD; 1 -145 residue) and a C-terminal domain (CTD; 151 -231 residue), connected by a 5-residue flexible linker. The structures of the full-length capsid [24-28], NTD [29] and CTD domains [30-32] have been studied by crystallography, cryo-electron microscopy and NMR. The NTD domain is composed of 7 a-helices (CA helices 1 - 7), while the smaller CTD domain is composed of a short 3 io helix followed by an extended strand and 4 a-helices (CA helices 8-1 1). In solution, HIV-1 CA dimerizes with a dissociation constant (Kd) of 18 μΜ. This dimerization is mainly dependent on Trpl 84 and Metl 85 residues in Helix 9 of the CTD [31]. Mutation of these residues interferes with CA assembly in vitro [27, 33] and abolishes viral infectivity [31 , 34]. This interface is therefore required for efficient assembly of both the mature and immature capsid lattice [35, 36].

The HIV viral capsid protein is emerging as an interesting target for the development of new antiviral agents (reviewed in [37] ). Several screens have been conducted to identify small- molecule compounds or peptides inhibiting HIV- 1 infection, i.e., virtual screens [38-40], assays based on fluorescence-based assembly with CA-NC fusion proteins [41 -43], cell-based single- cycle HIV infection [44], replicative infection [45], phage display against CA and CA-NC [46], peptide mimicking CTD helix 9 sequence [47] and more recently, bimolecular fluorescence complementation (BiFC) using CA-CTD protein [48]. These efforts resulted in the identification of compounds or peptides that target either the NTD domain: CAP-1 [39], PF-74 [49], BD and BM series compounds [42, 50], I-XW-053 [38], compound 48 [43] and BI-1 and BI-2 [44]; or the CTD domain: CAC-1 [47], CAI [46], NYAD-1 [51], NYAD-201/202 [52], NYAD-36/66/67 [53] and small organic compounds [40], Unfortunately, thus far, none of the most promising candidates is used in the clinic. Bevirimat, which blocks HIV-1 replication by interfering with the Gag CA-SP1 cleavage site, was tested in clinical trials [54, 55]. However, even though Bevirimat significantly reduced viral load in both ART-treated and naive patients, a high baseline drug resistance was observed due to natural viral polymorphisms [56, 57].

SUMMARY

The HIV-1 capsid protein is a target of interest for the development of antiretro viral therapeutics as it is of crucial structural importance for HIV-1 replication. This protein plays essential roles in both early (uncoating) and late (capsid assembly and maturation) events of the HIV-1 life cycle. Using a time-resolved fluorescence resonance energy transfer based high- throughput screening assay (HTS-TR-FRET) screening assay for the identification of inhibitors of CTD capsid dimerization, we screened the Library of Pharmacologically Active compounds (LOPAC library) and identified Ebselen (2-Phenyl-l,2-benzisoselenazol-3(2H)-one), an organoselenium compound, as a potent inhibitor of capsid dimerization and HIV-1 replication.

We confirmed the direct interaction of Ebselen with the HIV-1 capsid CTD by Nuclear magnetic resonance (NMR) spectroscopic, and demonstrated that this compound covalently binds the CTD, likely via a selenylsulfide linkage with two cysteines residues, CI 98 and C 218. We demonstrated that Ebselen presents an anti-HIV-1 activity in single- and multiple-round of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. This compound acts at an early, post-entry stage of the HIV-1 replication cycle, by targeting a step prior to reverse transcription. It impairs the uncoating process by stabilizing the conical capsid core. It also potently blocks infection of different retroviruses such as Moloney murine leukemia virus and Simian Immunodeficiency virus, but displays no inhibitory activity against Hepatitis C and Influenza viruses. Our efforts not only resulted in the identification of a novel class of compounds with antiretroviral activity but also validate the use of HTS-TR-FRET screens for the identification of inhibitors of HIV- 1 capsid dimerization, which further strengthen the notion that the HIV-1 capsid is a promising antiviral target for drug development.

Accordingly, the invention provides, in various embodiments, a method for the inhibition of assembly and disassembly of HIV-1 capsid, comprising contacting a cell or a population of cells infected with HIV-1 and an effective amount or concentration of a compound of formula (I)

wherein

ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X; ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;

Q is C(=0) or S0₂;

X is Se, Se(O), S, S(O), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (Cl-C4)alkoxy, (Cl-C4)haloalkoxy, halo, cyano, nitro, (Cl-C4)alkyl-S(0)_q wherein q = 0, 1 , or 2, (Cl -C4)alkyl-N(R)S0₂, (Cl -C4)alkyl-S0₂N(R), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl = 0, 1 , 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4; or a pharmaceutically acceptable salt thereof.

In various embodiments, the invention provides a method of treatment or inhibition of an HIV-1 infection in a human, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I).

In various embodiments, a compound suitable for carrying out a method of the invention is a compound of formula (IA)

wherein

X is Se, Se(O), S, S(O) or S(0)₂;

wherein the ring directly bonded to group X can further comprise one or two nitrogen atoms therewithin;

Y is CR or N;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (Cl-C4)alkoxy, (Cl -C4)haloalkoxy, halo, cyano, nitro, (Cl -C4)alkyl-S(0)_q wherein q = 0, 1 , or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-S0₂N(R), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl = 0, 1, 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4; or a pharmaceutically acceptable salt thereof.

In various embodiments, the invention provides a method of treatment or inhibition of an HIV-1 infection in a human, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (IA).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. CTD/CTD dimerization. (A) Schematic representation of TR-FRET assay. (B) Schematic representation of CTD protein constructs. (C) Analysis of purified CTD proteins by Coomassie blue staining and western-blot using anti-p24 antibody. (D) ELISA assay revealing GST-CTD/CTD-Flag interaction. The background controls in the assay contained buffer only, GST-CTD and CTD-Flag alone and GST incubated with CTD-Flag protein. (E) ELISA assay revealing inhibition of CTD dimerization by untagged CTD. A range of untagged CTD protein was added to constant concentrations of GST-CTD and CTD-Flag. The background controls in the assay contained buffer only, GST-CTD, CTD-Flag and untagged CTD alone. The data shown were representative of three independent experiments with triplicate data points in each experiment. (F) Time-resolved fluorescence-resonance energy transfer (TR-FRET) assay revealing GST-CTD/CTD-Flag dimerization. The background controls in the assay contained GST-CTD alone, anti-GST and anti-Flag antibodies alone and GST incubated with CTD-Flag protein. Untagged CTD at 100 μΜ concentration was used as inhibitor of CTD/CTD

dimerization. The transfer of fluorescence was measured as ratio of (signal at 665/signal at 620)* 10,000. The Signal-to-background (S:B) and Z' score are indicated. (G) TR-FRET assay revealing inhibition of CTD dimerization by untagged CTD. A range of untagged CTD protein was added to constant concentrations of GST-CTD and CTD-Flag.

Figure 2. LOPAC pilot screen. (A) Summary of 1536-well format assay protocol for TR-FRET assay used to screen the LOPAC library. (B) Primary results screen. Each black plot represents one compound screened in triplicate. Red plots (series at around 100 on "% Response" y-axis ) correspond to GST-CTD alone and represent the "no CTD dimerization" baseline. Green plots (series at around 0 to -10 on y-axis) correspond to GST-CTD and CTD-Flag interaction and represent the "100% dimerization" baseline. Blue plots (at around 70% on y-axis) correspond to GST-CTD and CTD-Flag incubated with 3 μΜ untagged CTD, which results in 75% inhibition of CTD dimerization. Statistics of the screen are indicated. (C) Structure of Ebselen compound. (D) Inhibition of CTD dimerization by Ebselen in TR-FRET assay. The data corresponds to the mean of four independent experiments with triplicate data points in each experiment.

Figure 3. Activity of Ebselen on HIV-1 replication. (A) HeLa-CD4-LacZ cells were infected with NL4.3 replicative virus in the presence of increasing concentrations of Ebselen for 72 hrs. Beta-galactosidase activity was determined by quantitative CPRG assay. (B) Viral supernatants from Hela-CD4 infection were assayed for their p24 antigen content using a sandwich ELISA kit. (C) Viral supernatants from PBMCs infection were assayed for their p24 antigen content using a sandwich ELISA kit. (D) Cell viability MTT assay on HeLa-CD4 cells treated with increasing concentration of Ebselen for 72 hrs. (E) Cell viability MTT assay of PBMCs treated with increasing concentration of Ebselen for 4 days.

Figure 4. Ebselen induces local structural changes in CA-CTD. (A-B) 2D ['H- ^NJ-HSQC spectra of CA-CTD with 1.2- (A) or 2.4-fold (B) molar excess of Ebselen. (C-D) 2D [Ή-¹⁵Ν]- HSQC spectra of CA-CTD W184A/M185A (WAMA) with 1.2- (C) or 2.4-fold (D) molar excess of Ebselen. Peak assignments were based on published HSQC chemical shifts for CA-CTD [58] and the WAMA mutant [59]. (E) Circular dichroism of CA-CTD with or without 2.4x Ebselen. (F) Tryptophan fluorescence quenching assay of CA-CTD or lysozyme as negative control, titrated with Ebselen or Raltegravir. Control is black in gray-scale, experiment is light in gray scale.

Figure 5. Ebselen covalently binds to both CA-CTD Cys residues. (A) Representation of frequencies of C198 and C218 residues from 2890 sequences from 8 different clades (A, B, C, D, F, G, CRF01_AE and CRF01_AG) (HIV sequence database alignment) using the WebLogo application (http://weblogo.berkeley.edu). (B) ESI-MS analysis of CA-CTD incubated with different molar ratios of Ebselen (Ebs). (C) ESI-MS analysis of oxidized CA-CTD species (intramolecular disulfide bridge, or higher-order cysteine oxidations) treated with different molar ratios of Ebselen.

Figure 6. Reversibility of Ebselen-CA-CTD interaction. Effect of DTT (5 mM) post- incubation on Ebselen-CA-CTD interaction analyzed by tryptophane fluorescence assay (A) and LC-ESI-MS (B).

Figure 7. Model of CA-CTD (Ebselen)₂ binding. Ribbon model of CA-CTD monomer (PDB ID: 2BUO) rendered in PyMOL 11 Hybrid, with Cys 198 and Cys218 (magenta) linked via selenylsulfide bonds to two molecules of Ebselen. The ligands are shown as sticks, with C, N, O, Se colored in green, blue, red and yellow, respectively.

Figure 8. Residues affected by Ebselen in ^I5N-CA-CTD. (A) HSQC peak shift with respect to CA-CTD (black) and WAMA (red) apo after adding 1.2: 1 molar excess of Ebselen. (B-C) HIV capsid CTD structure (PDB 2BUO) rendered in PyMOL as a ribbon

diagram highlighting residues affected by adding 1.2: 1 molar excess of Ebselen to CA-CTD (B) and WAMA (C). Residue color scheme reflects peak shifts that are > +2 SD (red sticks), between +1 to +2 SD (orange) and within +1 SD from the mean peak perturbation (light orange). Residues Cys 198 and Cys 218 are shown as magenta sticks.

Figure 9. Ebselen covalent linkage results in CTD dimer dissociation. (A) Overlay of CA- CTD (black) and WAMA (red) HSQC spectra, where peaks that are substantially broadened in WAMA are labeled in CA-CTD; peaks that are mutated to alanine, namely W184 and Ml 85, are also labeled. (B) Ribbon diagram of CA-CTD (PDB 2BUO) where peaks that are perturbed after adding 2.4: 1 Ebselen, but not by 1.2: 1 Ebselen are colored in red; residues in the dimerization domain are rendered as sticks. Cys 198 and Cys 218 are shown as magenta sticks. (C) Similar ribbon diagram as in (B), where residues corresponding to peaks in CA-CTD that disappeared in WAMA are colored in red, while residues in the dimerization domain are shown as sticks. The sites of W184A and M185A substitutions are shown as cyan sticks and Cys 198 and Cys 218 are rendered as magenta sticks.

Figure 10. Mechanism of action of Ebselen. (A-C) Impact of Ebselen on early replication events (A) Activity of Ebselen on HIV-1 single-round infection in HeLa-CD4-LacZ cells. Beta- galactosidase activity was determined by quantitative CPRG assay. (B) Effect of Ebselen on reverse-transcription products and HIV-1 integration. Early and late RT products were determined by qPCR, and provirus integration was quantified by Alu-PCR followed by a qPCR. (C) Impact of Ebselen on HIV-1 capsid stability by fate of capsid assay. (D) Impact of Ebselen on in vitro capsid assembly. (E-G) Impact of Ebselen on late viral replication events. (E) Effect of Ebselen on Tat-mediated transactivation in Hela-CD4-LTR-luc. Luciferase activity was measured 48 hrs post-transfection of Tat-Flag. (F) Activity of the indicated drugs on Gag expression and maturation in 293T cells transfected with pNL4.3. Gag expression quantified by western blot with corresponding densitometry analysis (bottom). (G) Impact of Ebselen on infectivity of the viral particles produced in (F) in infection of the reporter Hela-CD4-LTR-Luc cells. Luciferase activity measured 48 h post-infection.

Figure 11. Impact of Ebselen on MoMuLV, SIV, HCV and Influenza viruses. (A) Impact of Ebselen on MoMuLV retrovirus in TE671 cells infected with VSV-G-MoMuLV-GFP and VSV- G-NL4.3-GFP viruses. The percentage of GFP positive cells was determined by flow cytometry. (B) Impact of Ebselen on SIV infection in CEM174 cells infected with SIVmac239 virus. Viral supernatants were assayed for their p27 antigen content using a sandwich ELISA kit (C) Impact of Ebselen on HCV virus by replication and infectivity assays in Huh-7.5 cells containing an HCV reporter virus (pSG-Rluc-2a-neo-NS3-5B JFH1) by measuring luciferase activity (left) or infected with a cell-culture adapted strain of JCl (JCl . l) by determining the TCID₅o/mL using an anti-NS5A antibody. (D) Impact of Ebselen on Influenza virus (H1N1) in Hela-CD4 cells determined by flow cytometry using a monoclonal antibody to H1N1. DETAILED DESCRIPTION

Tables

HIV-1 virus subtype Coreceptor usage % inihibtion laboratory-adapted

pNL4.3 X4 88.7 (±7,7)

Primary isolates

92UG037 R5 90.4 (±8,8)

92RW00S A as 86.2 (±9,4)

92HTS96 B R5X4 97.1 (±1,9)

92US657 B R5 95.0 (±6,1)

93IN 101 C R5 86.8 (±8.0)

97ZA003 C R5 87.7 (±7.7)

92UG001 D R5X4 89.4 (±8,0)

94UG118 O R5 97.2 (±2.7)

93TH051 AE R5X4 93.0 (±2.0)

CMU02 AE X4

93BR020 F R5X4 96.2 (±3,5)

63 6 R5 88.8 (±10.0)

RU570 6 R5 98.6 (±1.2) drug-resistant virus

AZT-resistant B X4 97.4 (±1.6)

Nevirapine-resistant B X4 94.2 (±3.9)

Table 1 % of CTD

compound structure dlmerization EC._j0 (μΜ) CC₅₀ (μΜ) inhibition

Ebsefen 65,8 ±1.6% 0.99 ±0.22 32.7 ±0.06

Omeprazole 34.7 i 14.1 % > 10 > 50

Methy|.3,4.<Jephostatl 41.8 ± 1.6% > 10 31.3 ± 2.07

o

3-Bromo-l,l,l- _R jj . _{> 1Q > SQ} trifluoroaeetone (BTFA) ^*\/^-Q

Table 2 % of CTD

compound structure dlmerlzation IC_M (μ ) EC M) CC^ {μΜ|

Inhibition 7.3

benzt50seler»azot-3{2H)-one}

Ebstten Se!enox!d*

{i-oxkh?-2-Phenyi-.,2- 81*4.1% 0.0512 1.61+048 28.9 ± 3.3

Table 3 Table 1. Ebselen inhibits HIV-1 primary isolates. Inhibitory activity of Ebselen against diverse primary isolates and drug resistant strains was determined in TZM-bl cells in presence of 10 μΜ Ebselen.

Table 2. Structure and activity of cysteine-binding compounds. Maximal % inhibitory activity of cysteine-binding compounds was measured in TR-FRET assay at a final concentration of 10 μΜ. IC₅o of analogs was determined by a dose-response inhibition of CTD dimerization in TR-FRET assay. ECso was determined in HIV-1 single-round infection in Hela-CD4-LTR-LacZ cells. CC50 were determined by an MTT assay on HeLa-CD4-LTR-LacZ cells for a period of 48 hrs.

-: inhibition by less than 25%

Table 3. Structure and activity of Ebselen analogs. Maximal % inhibitory activity of Ebselen and analogs was measured in TR-FRET assay at a final concentration of 10 μΜ. IC₅o of analogs was determined by a dose-response inhibition of CTD dimerization in TR-FRET assay. EC50 of Ebselen, 2pyr-Ebselen and Ebselen oxide was determined in HIV-1 single-round infection in Hela-CD4-LTR-LacZ cells. CC50 were determined by an MTT assay on HeLa-CD4-LTR-LacZ cells for a period of 48 hrs.

-: inhibition by less than 25%

ND: non determined

Description

The invention is directed, in various embodiments, to methods for interfering with assembly and disassembly of the capsid for the HIV-1 virus, through interference with the key step of dimerization and further associations of capsid CA protein. The compound covalently binds to the C-terminal domain of the viral capsid, domain that plays an essential role in the formation of the intact viral capsid structure, necessary for the formation of an infectious viral particle. The invention is consequently directed to methods for the inhibition and the treatment of HIV-1 infection in humans.

The invention provides, in various embodiments, a method for the inhibition of assembly and disassembly of HIV-1 capsid, comprising contacting a cell or a population of cells infected with HIV-1 and an effective amount or concentration of a compound of formula (I)

wherein

Q is C(=0) or S0₂;

X is Se, Se(O), S, S(O), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (Cl-C4)alkoxy, (Cl-C4)haloalkoxy, halo, cyano, nitro, (Cl -C4)alkyl-S(0)_q wherein q = 0, 1, or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-S0₂N(R), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl = 0, 1 , 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4; or a pharmaceutically acceptable salt thereof. In various embodiments, a compound suitable for carrying out this method is a compound of formula (IA)

wherein

X is Se, Se(O), S, S(O) or S(0)₂;

Y is CR or N;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl); J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (Cl-C4)alkoxy, (Cl-C4)haloalkoxy, halo, cyano, nitro, (Cl-C4)alkyl-S(0)_q wherein q = 0, 1, or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-S0₂N(R), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl = 0, 1, 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4;

or a pharmaceutically acceptable salt thereof,

In various embodiments, a specific compound suitable for carrying out this method can be of any one of the following formulas:

or a pharmaceutically acceptable salt thereof.

In various embodiments, the invention provides a method of treatment or inhibition of an HIV-1 infection in a human, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)

wherein

Q is C(=0) or S0₂;

X is Se, Se(O), S, S(O), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (Cl-C4)alkoxy, (Cl-C4)haloalkoxy, halo, cyano, nitro, (Cl-C4)alkyl-S(0)_q wherein q = 0, 1 , or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-S0₂N(R), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl = 0, 1, 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4; or a pharmaceutically acceptable salt thereof; In various embodiments, a compound suitable for carrying out this method is a compound of formula (IA)

wherein

X is Se, Se(O), S, S(O) or S(0)₂;

Y is CR or N;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl); J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl - C4)haloalkyl, (Cl-C4)alkoxy, (Cl-C4)haloalkoxy, halo, cyano, nitro, (Cl-C4)alkyl-S(0)_q wherein q = 0, 1, or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-S0₂N(R), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl = 0, 1 , 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4;

or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof.

The invention further rovides a medical use of a compound of formula (I)

wherein ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X; ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;

Q is C(=0) or S0₂;

X is Se, Se(O), S, S(O), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

nl = 0, 1 , 2, 3, or 4; n2 = 0, 1, 2, 3, or 4; or a pharmaceutically acceptable salt thereof; for treatment or inhibition of an HIV-1 infection in a human patient.

Furthermore, a compound of formula (I) can be used for the preparation of a medicament for the treatment or inhibition of HIV-1 infection in a human patient.

More specifically, for these uses, the compound can be of formula (IA)

wherein

X is Se, Se(O), S, S(O) or S(0)₂;

Y is CR or N;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (Cl-C4)alkoxy, (Cl-C4)haloalkoxy, halo, cyano, nitro, (Cl-C4)alkyl-S(0)_q wherein q = 0, 1 , or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-S0₂N(R), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(==0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl - C4)alkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl = 0, 1 , 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4;

or a pharmaceutically acceptable salt thereof; or can be of any one of the following formulas:

or a pharmaceutically acceptable salt thereof.

The invention further provides a compound of formula (I)

wherein

ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X; ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof; Q is C(=0) or S0₂;

X is Se, Se(O), S, S(O), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl -C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (C 1 -C4)alkoxy, (Cl-C4)haloalkoxy, halo, cyano, nitro, (Cl-C4)alkyl-S(0)_q wherein q - 0, 1 , or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl -C4)alkyl-S0₂N(R), (Cl -C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl = 0, 1 , 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4; or a pharmaceutically acceptable salt thereof; wherein the compound is not ebselen.

In various embodiments, the invention provides a compound of formula (IA)

wherein

X is Se, Se(O), S, S(O) or S(0)₂;

Y is CR or N;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl - C4)haloalkyl, (C 1 -C4)alkoxy, (Cl-C4)haloalkoxy, halo, cyano, nitro, (Cl -C4)alkyl-S(0)_q wherein q = 0, 1 , or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-S0₂N(R), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(=0)N(R), or (Cl -C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted; nl = 0, 1 , 2, 3, or 4; n2 = 0, 1 , 2, 3, or 4;

or a pharmaceutically acceptable salt thereof;

wherein the compound is not ebselen.

The invention further provides a compound of any of the following formulas:

or a pharmaceutically acceptable salt thereof.

The CTD domain of CA is responsible for capsid dimerization, and Trpl 84 and Metl 85 residues in helix 9 of the CTD mediate this interaction [31]. A peptide mimicking the helix 9 sequence, CAC-1 , was shown to inhibit CTD-CTD dimerization [47], and a stabilized version of this peptide, NYAD-201 , displayed inhibitory activity on HIV-1 replication and inhibited in vitro capsid assembly [52]. These results suggested that targeting CTD dimerization would successfully lead to the identification of potent HIV inhibitors. Thus, we developed a primary HTS based on TR-FRET assay using the CTD domain of CA tagged with either GST or Flag (Fig. 1A).

HIV-1 CTD proteins were expressed as fusion proteins with either GST in N-terminal (GST-CTD) or Flag in C-terminal (CTD-Flag), both tagged with 6 histidines in N-terminal (Fig. IB), Free CTD domain was expressed and used as control for inhibition of CTD dimerization. The proteins were produced in E.coli and purified by affinity chromatography on a Ni-NTA agarose column. The identity and homogeneity of the proteins were verified by SDS-PAGE followed by Coomassie blue staining and western-blot, revealing a major expected band at 37 kDa for GST-CTD, 12 kDa for CTD-Flag and 1 1 kDa for CTD (Fig. 1C).

CTD dimerization and its inhibition monitored by ELISA and TR-FRET

First, to validate the GST-CTD/CTD-Flag dimerization, we performed an ELISA, by coating GST-CTD protein onto a 96-well plate and adding a range of CTD-Flag protein concentrations. The interaction was revealed using an anti-Flag antibody followed by TMB substrate. A dose-dependent signal was observed, revealing the dimerization of CTD capsid (Fig. ID). Free GST was used as negative control. When increasing concentrations of untagged CTD domain were added, the GST-CTD/CTD-Flag dimerization was inhibited with an EC₅o of 1.9 ± 0.12 μΜ (Fig. IE).

To screen a small molecule compound library, we developed an HTS assay. As such, CTD dimerization was assessed by TR-FRET using the donor europium (Eu) cryptate- conjugated anti-GST and the acceptor allophycocyanin (XL-665)-conjugated anti-Flag antibodies. In this system, emission at 665 mm would reflect the interaction of CTD proteins. A strong and specific absorbance was obtained between GST-CTD and CTD-Flag, while the interaction between free GST and CTD-Flag gave a background signal (Fig. IF). The CTD dimerization was inhibited by a range of untagged CTD concentrations with an EC50 of 1.07 μΜ (Fig. 1G). This assay presented excellent statistics with a Z' score > 0.7 and a signal-to- background ratio of 3.5, allowing to proceed with screening of the LOP AC.

LOPAC screen

The HTS-TR-FRET assay was miniaturized and implemented in a 1536-well plate format following the protocol in Figure 2A. The LOPAC library, composed of 1,280 pharmacologically active approved small molecules, was screened in triplicate at a concentration of 10 μΜ. The results were summarized as a scatterplot with black plots representing individual compounds of the LOPAC library. GST-CTD protein alone was used as high control corresponding to "100% inhibition" (red plots) while GST-CTD/CTD-Flag proteins were used as low control

corresponding to "100% CTD dimerization" (green plots). Untagged CTD at 3 μΜ concentration was included in every plate as control and achieved 75% inhibition of CTD dimerization (blue plots) (Fig. 2B). The LOPAC screen was completed with excellent assay statistics between triplicates with Average Z' = 0.890 ± 0.004; Average Z = 0.573 ± 0.003; S:B = 5.629 ± 0.101 (Fig. 2B). The hit cutoff was set at 42% (AVEG + 3SD) and the hit rate was 3.28%

corresponding to 42 compounds. The top 10 hit compounds inhibited CTD-CTD interaction by 71% to 59%). The LOPAC library was cross-referenced for HCV core inhibitors [60] and out of the 42 hits identified in our screen, 9 displayed inhibitory activity against HCV core dimerization (15.7 % to 51.6 %). We tested 20 of the top hits in multiple round of HIV- 1 infection in HeLa- CD4 cells and identified Ebselen, an organoselenium compound (Fig. 2C), as the most potent inhibitor of HIV- 1 replication. The inhibitory effect of Ebselen on CTD dimerization was confirmed by TR-FRET with Ebselen displaying an IC50 of 46.1 nM (Fig. 2D). In comparison, Ebselen presented no activity on HCV core CI 06 dimerization by TR-FRET assays [60].

Ebselen inhibits HIV-1 replication

We evaluated HIV-1 susceptibility to Ebselen using a reporter cell line that stably expresses the β-galactosidase (LacZ) gene driven by the HIV-1 5' long terminal repeat (LTR) and responds to the viral protein Tat expressed by an incoming virus. Thus, HeLa-CD4-LTR- LacZ cells were infected with the HIV isolate NL4.3 in the presence of increasing concentrations of Ebselen, and the measured β-gal activity was directly correlated with viral replication. Ebselen inhibited the infection in a dose-dependent manner with an EC50 of 1.99 ± 0.57 μΜ (Fig. 3A), while Efavirenz, a reverse transcriptase inhibitor (NNRTI) presented an EC50 of 1.02 ± 0.05 nM, and Lopinavir, a protease inhibitor (PI) showed maximal inhibition of only 30%. Given that Pis inhibit capsid maturation and only act upon spreading from the initial infection, it was not surprising to observe Lopinavir's low inhibitory activity in this 72-hour assay. The activity of Ebselen on HIV-1 replication was also assessed by p24 ELISA in the supernatant, with an EC₅₀ of 7.2 μΜ in Hela-CD4-LTR-LacZ (Fig. 3B) and 3.2 ± 0.9 μΜ in peripheral blood mononucleated cells (PBMCs) (Fig. 3C). Given that Ebselen is expected to bind to capsid, we made sure Ebselen was not interfering with the p24 detection by ELISA. The concentration of Ebselen used in these assays did not impact cell viability as Ebselen displayed a half-maximal cytotoxic concentration (CC₅₀) of 25.4 ± 2.9 μΜ in HeLa-CD4-LTR-LacZ (Fig. 3D) and > 30 μΜ in PBMCs (Fig. 3E). We then analyzed the antiviral activity of Ebselen against thirteen HIV- 1 primary isolates belonging to seven different clades and two drug-resistant viruses, in presence of 10 μΜ Ebselen in TZM-bl cells. TZM-bl are Hela-CD4 cells that express the receptor (CD4) and the two coreceptors (CXCR4 and CCR5) of HIV-1, and express the β-galactosidase and luciferase genes under control of the HIV- 1 promoter. In this reporter system, Ebselen displayed between 86.2 and 98.6% inhibitory activity against all viruses (Table 1).

Ebselen covalently links with Cys residues in the CTD of capsid

We monitored the binding of Ebselen to CTD via [^!H,¹⁵N]-heteronuclear single quantum coherence (HSQC) NMR on CA-CTD and on the capsid mutant CA-CTD W184A/M185A (WAMA) that bears amino acid substitutions that disrupt dimerization [31]. Peak assignments were based on published HSQC chemical shifts for CA-CTD [58] and the WAMA mutant [59]. The addition of 1.2- and 2.4-fold molar excess of Ebselen to CA-CTD and WAMA revealed peaks corresponding to free CA-CTD but with lower intensity relative to apo and the concomitant appearance of new resonances from ligand-bound protein shifted from initial peak positions, consistent with ligand binding in slow exchange on the NMR timescale (Fig. 4A-D). After adding 2.4x Ebselen to either protein, high-intensity peaks clustered at the spectral center was observed, indicating protein aggregation (Fig. 4B and 4D). Circular dichroism (CD) spectrometry showed identical spectra for the free and Ebselen-saturated protein (Fig. 4E), suggesting that the aggregated protein observed at 2.4x Ebselen did not result from protein unfolding, but from structural rearrangement to accommodate the bulky and hydrophobic ligand, resulting in a conformation susceptible to aggregation.

Binding of Ebselen to CA-CTD was also confirmed by tryptophan fluorescence quenching, showing a decrease in fluorescence proportional to increased Ebselen concentrations (Figure 4F). No fluorescence quenching was observed in the presence of the integrase inhibitor (IN), Raltegravir, or when Ebselen was incubated with lysozyme, demonstrating the specificity of the Ebselen:CA-CTD interaction. Furthermore, the addition of dithiothreitol (DTT) to Ebselen-CA- CTD resulted in a loss of fluorescence quenching suggesting that the interaction between Ebselen and CA-CTD was reversible by a reducing agent (Fig. 6A).

Ebselen is an organoselenium compound that mimics glutathione peroxidase activity (review in [61]). Previous studies show that this compound forms a covalent bond between its selenium atom and thiols in cysteine residues by forming a selenylsulfide (-Se-S-) linkage [62-64], HIV-1 capsid contains 2 cysteine residues, Cysl98 and Cys218 (full-length CA number), both located in the CTD. These cysteines residues are highly conserved across HIV-1 subtypes with 99.96% and 99.76% conservation for CI 98 and C218, respectively (Fig. 5 A). These cysteines do not form a disulfide bond in the viral particle, and mutation of Cysl98 interferes with viral particle disassembly, while mutation of Cys218 drastically reduces viral particle assembly [65], Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) confirmed the covalent linkage of Ebselen to CA-CTD (Fig. 5B). Incubation of CA-CTD with equimolar amount of Ebselen showed the presence of intact CA-CTD (-10,838 Da) as well as ion masses that are 274 Da and 548 Da higher than CA-CTD, consistent to mass increases of one or two covalently linked Ebselen molecules (-274 Da). Notably, CA-CTD became preferentially double labeled with two molecules of Ebselen even at a compound-to-protein molar ratio of 1 : 1. At higher Ebselen to CA-CTD ratios only the doubly modified CA-CTD was detected. These observations highlight the high reactivity of both cysteine residues for Ebselen modification. In accordance with the tryptophan fluorescence quenching results, we confirmed that addition of DTT reduced the selenylsulfide bond between Ebselen and CA-CTD (Fig. 6B). Furthermore, low amounts of CA-CTD protein isoforms with intramolecular disulfide-linked (Cys-S-S-Cys) and oxidized Cys residues (Cys-S0₂H) were also observed, but not the ion species equivalent to the corresponding Ebselen-linked oxidized CA-CTD (Fig. 5C). Collectively, these results suggest that Ebselen covalently links to thiol groups (Cys-SH) in Cysl98 and Cys218 of CA-CTD via a selenylsulfide linkage, forming S-[(2-carbamoylphenyl)selanyl]-L-cysteine, as illustrated in the structural model of CA-CTD bound to two Ebselen molecules (Fig. 7). The binding of Ebselen to CTD is specific as Omeprazole [66], Methyl-3,4-dephostatin [62] and 3-Bromo- 1 , 1 ,1 - trifluoroacetone (BTFA) [67], three cysteine-binding compounds, have no effect on HIV-1 replication and display less or no activity on CTD dimerization inhibition compared to Ebselen (Table 2).

NMR chemical shift perturbations in CA-CTD and WAMA in the presence of 1.2-fold molar excess of Ebselen were quantitatively analyzed in a plot overlay that showed similar perturbation footprint (Fig. 8A). We mapped the residues that were affected by Ebselen in CA-CTD and WAMA to the crystal structure of CA-CTD [68], and the most perturbed residues were found to co-localize in close proximity to Cysl 98 and Cys218. Some residues distal from these cysteine targets were also perturbed allosterically as a result of either structural rearrangements or conformational change after Ebselen linkage (Fig. 8B and 8C). At 2.4-fold molar excess of Ebselen, additional residues in CA-CTD were perturbed that coincides with peaks that were substantially broadened in WAMA relative to CA-CTD (Fig. 9). These peaks correspond to several critical residues within or in close proximity to the dimerization region, including W184 and Ml 85. These results indicate that covalent attachment of two molar equivalents of Ebselen promotes a conformational change that shifts the monomer-dimer equilibrium in favor of the Ebselen-linked monomer that is susceptible to aggregation.

Mechanism of action of Ebselen

Capsid inhibitors as well as restriction factors targeting the viral capsid can inhibit HIV-1 replication at different steps of the viral life cycle, such as post-entry events (uncoating), capsid assembly or capsid maturation. We investigated in detail the step of HIV-1 life cycle inhibited by Ebselen, starting with the analysis of the activity of Ebselen in early stages of HIV-1 replication (post-entry events) in single-round infection using VSV-G pseudotyped virus (VSV-G-NL4-3). Ebselen was active in single-round infection assay with an EC₅₀ of 3.37 ± 0.82 μΜ. Efavirenz displayed an EC50 of 1.22 ± 0.22 nM, while Lopinavir, as expected, was not active in this assay (Fig. 10A). We subsequently analyzed the viral DNA species, such as the early and late reverse transcription (RT) products, via quantitative PCR (qPCR). In addition, we also analyzed proviral integration by Alu-PCR followed by qPCR (Fig. 10B). Ebselen decreased early and late RT products in a dose-dependent manner, and as a result, inhibited the levels of integrated proviral DNA. Raltegravir (IN) and Lopinavir (PI) had no impact on RT activity, and as expected Raltegravir inhibited proviral integration. The profile of Ebselen was similar to that observed for the RT inhibitor Efavirenz (NNRTI), which suggests Ebselen may affect the reverse transcription process by negatively impacting the uncoating process. To test this hypothesis, we investigated the fate of the HIV-1 capsid in the presence of Ebselen. This assay monitors the uncoating process by measuring the relative level of soluble (disassembled CA) vs pelletable (fullerene core) capsid during HIV-1 infection [69]. As shown in Figure IOC, Ebselen increased the amount of pelletable CA compared to DMSO control, suggesting a stabilization of the mature capsid, similar to what is observed for the cellular restriction factor MxB [14]. In contrast, the NTD inhibitor PF74 decreased the amount of pelletable CA protein during infection by destabilizing the mature capsid, as previously observed [70]. The stabilization effect observed in presence of Ebselen was also shown in in vitro capsid multimerization assay. While the addition of CTD and CAI (capsid assembly inhibitor) peptide inhibited capsid assembly [33, 71], addition of increasing concentration of Ebselen resulted in a dose-dependent increase in the rate of CA multimerization. This same phenomenon was previously observed with the NTD capsid inhibitor PF74 [49]. In contrast, the addition of Raltegravir (IN) had no impact on HIV-1 capsid assembly (Fig. 10D). Altogether, these results suggest that Ebselen acts at an early, post-entry stage of the HIV-1 replication cycle, by increasing the stability of the viral capsid during infection, and impairing the uncoating process by a mechanism of action different from PF74. We cannot exclude the possibility that this increase in stability may result from aggregation of CTD protein as observed in NMR in presence of 2.4-fold excess Ebselen.

We further investigated the impact of Ebselen on late replication events (after integration of the viral DNA into the host cell genome). First we analyzed the impact of Ebselen on HIV transcription promoted by the viral transactivator, Tat, using Hela-CD4-LTR-Luc cells that respond to Tat transactivation (Fig. 10E). We demonstrated that Ebselen had no impact on the ability of Tat to activate HIV-1 transcription, while dCA, a potent Tat inhibitor [72], displayed an inhibitory activity of 70%. We then analyzed the impact of Ebselen on Gag Expression and maturation after transfection of HEK-293T cells with pNL4.3 as well as on the infectivity of the viral particles produced. The Gag protein expression and maturation was similar in presence of Ebselen, Raltegravir (IN) or DMSO control, while as expected, two protease inhibitors (Ritonavir and Lopinavir) affected the maturation process (Fig. 10F). The viral particles produced in presence of Ebselen and Raltegravir showed similar infectivity to the virions produced in DMSO control, while Ritonavir and Lopinavir abolished the infectivity of the virions by interfering with the maturation process (Fig. 10G). Taken together, these results demonstrate that Ebselen has no impact on late events in the HIV-1 life cycle.

Evaluation of analogs of Ebselen

Based on our NMR and LC-ESI-MS results, and as reported in the literature [62-64], Ebselen forms a selenylsulfide (-Se-S-) linkage with cysteine residues. To confirm the importance of the selenium atom in Ebselen, we analyzed analogs with selenium substituted by sulfur (S-Ebselen), oxygen (O-Ebselen), carbon (C-Ebselen) [73] or a selenoxide group (Ebselen oxide), or with the phenyl ring converted to pyridine (2pyr-Ebselen and 3pyr-Ebselen) [74] (Table 3). The inhibitory activity of these analogs on CTD dimerization was evaluated by TR- FRET assay. Only S-Ebselen, 2pyr-Ebselen and Ebselen oxide analogs retained the inhibitory activity of Ebselen, while O-Ebselen, C-Ebselen and 3pyr-Ebselen had no impact on dimerization. Since Ebselen oxide and 2pyr-Ebselen displayed an IC50 in the same range as Ebselen in TR-FRET assay, we further studied these compounds in single-round infection and determined their cytotoxicity. Ebselen oxide and 2pyr-Ebselen presented a slightly lower EC50 than Ebselen (1.61 ± 0.18 μΜ and 1.92 ± 0.85 μΜ vs 4.07 ± 0.93 μΜ, respectively); however Ebselen oxide was slightly more toxic than Ebselen in HeLa-CD4 cells (CC₅₀ = 28.9 ± 3.3 μΜ vs 37.9 ± 7.3 μΜ). Taken together, Ebselen modification to Ebselen oxide or 2pyr-Ebselen slightly improved its potency. Collectively, these results reveal the importance of Ebselen' s selenium atom in its antiviral activity.

Impact of Ebselen on other retroviruses and non-retroviruses

We assessed the specificity of Ebselen to inhibit infection by Moloney Murine Leukemia (MoMuLV) and Simian Immunodeficiency (SIVmac239) viruses. Ebselen inhibited MoMuLV and HIV-1 with an EC₅₀ of 3.87 μΜ and 1.75 μΜ, respectively (Fig. 1 1A), and SIV with an EC₅₀ of 4.2 μΜ (Fig. 1 IB). The inhibition of SIVmac239 by Ebselen was expected since SIV CTD sequence is relatively conserved to HIV-1 and also contains the two cysteine residues, Cysl 98 and Cys218 [75], However, little homology exists between MoMuLV and HIV-1 capsids. MoMuLV capsid contains a single cysteine residue at position 55 and a unique carboxy terminus containing charge-rich residues (arginine and glutamic acid), critical for proper viral assembly [76, 77]. These results suggest that the binding of Ebselen to capsid may also have a conformational component.

We further evaluated the effect of Ebselen on non-retroviruses such as Hepatitis C (HCV) and Influenza viruses. The ability of Ebselen to inhibit HCV infection was evaluated by replication and infectivity assays in Huh-7.5 cells containing an HCV reporter virus (pSG-Rluc-2a-neo- NS3-5B JFH1) or infected with a cell-culture adapted strain of JC1 (JC1.1). These cells were treated with two different concentrations of Ebselen, Raltegravir, BMS-052 (an active compound targeting the HCV NS5A protein, which disrupts both viral replication and assembly) or DMSO. While the HCV specific BMS-052 compound was shown to inhibit HCV replication and infectivity, Ebselen, as well as Raltegravir, presented no effect on HCV infection (Fig. 1 1C). The impact of Ebselen on Influenza virus was evaluated using H1N1 strain. The virus was mixed with Ebselen, Raltgravir or with DMSO as control and used to infect 293T cells. After 16 hrs post-infection, cells were fixed, stained with an anti-hemagglutinin antibody and the percentage of positive cells was determined by flow cytometry. Ebselen and Raltegravir, displayed no inhibition of H1N1 infection (Fig. 1 ID). These results suggest Ebselen to be rather specific for retroviruses. In a recent report, Ebselen was shown to inhibit HCV NS3 helicase binding to nucleic acid by interacting with cysteine residues, with an EC₅o = 30 ± 17 μΜ and a CC₅₀ = 34 ± 4 μΜ. Thus, the inhibitory activity of Ebselen could partly be due to cellular toxicity [78].

Conclusions

Protein-protein interactions play key roles in a wide range of biological processes, and are attractive targets for the design of novel therapeutics [79]. However, the development of inhibitors that target these interactions is far more challenging compared to enzyme active sites. Notably, none of the drugs currently used in antiretroviral therapy target the HIV-1 capsid protein. Given its key importance in productive infection, it seems reasonable to promote drug development against the capsid. HIV is well known for its high mutation rate and consequently, rapid site-directed drug resistance; thus developing inhibitors insensitive to such viral mutational evolution is important. Targeting the viral capsid seems appropriate since this protein has been shown to mutate less readily and presents 70% of sequence conservation between isolates [80]. To this end, we conducted a TR-FRET based HTS assay to identify inhibitors of HIV-1 capsid dimerization. This screening successfully identified Ebselen as an inhibitor of capsid dimerization and HIV-1 replication. NMR confirmed perturbation of residues in the dimerization domain of CTD when Ebselen is at more than two-fold the CTD molar concentration. Using cellular assays, we observed that this small molecule acts on the early post-entry step of the viral cycle prior to reverse transcription by stabilizing the incoming viral capsid. However, we cannot exclude the possibility that this increase in stability may result from aggregation of CTD protein as observed in NMR in presence of 2.4-fold excess Ebselen. Moreover, Ebselen was initially found as an inhibitor of dimerization in TR-FRET assays using only the CTD of capsid. In this context it is also a possibility that aggregation might have lead to a loss in fluorescence intensity. Interestingly, Ebselen inhibits diverse retroviruses but has no impact on non-related viruses such as HCV and influenza viruses. In addition, the selenium atom in Ebselen was shown to be important for its activity. However, Ebselen has been identified in several biological assays, suggesting lack of selectivity (reviewed in [61]), therefore chemical innovation around the Ebselen pharmacophore, while retaining the key selenium atom, will be necessary for increasing specificity for retroviral inhibition. A Ph2 clinical trial testing Ebselen (SPI-1005) to prevent and treat noise-induced hearing loss has been conducted. Ebselen demonstrated clinically relevant reduction in temporary threshold shift (TTS) induced by loud sound exposure and the doses of Ebselen administrated (200, 400 and 600 mg twice daily for 4 days) were well tolerated. Therefore, despite its lack of specificity, Ebselen is safely tolerated [81].

This study is a proof-of-concept employing a TR-FRET based HTS platform to identify a small molecule that targets the viral capsid. The identification of Ebselen as an inhibitor of HIV-1 replication, further highlights the validity of the HIV-1 capsid as a promising drug target. Moreover, as the use of covalent inhibitors is growing in drug discovery (reviewed in [82]), this study lays the groundwork for future drug design involving covalently-linked capsid inhibitors that target HIV-1 replication.

Materials and methods

Cells.

HeLa-CD4 cells expressing β-galactosidase (HeLa-CD4-LTR-LacZ) or Firefly luciferase (HeLa-CD4-LTR-Luc) genes under the control of HIV-1 LTR promoter were provided by Dr. Uriel Hazan, Universite de Cachan, France. HEK293T and the human rhabdomyosarcoma cell line TE671 were obtained from the American Type Culture Collection (ATCC). Human hepatocarcinoma cell line Huh7.5 cells were provided by Dr. Charles Rice of Rockefeller University. Human peripheral blood mononuclear cells were obtained from the blood of healthy seronegative donors.

HeLa, HeLa-CD4-LTR-Luc, HeLa-CD4-LTR-LacZ, HEK293T and TE671 cells were cultured in DMEM supplemented with 5% FBS, L-glutamine (292 μg/mL) and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37°C and 5% C0₂. Huh-7.5 cells were cultures in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin antibiotics and 1% of non-essential amino acids (NEAA).

Compounds: LOPAC library, Ebselen and analogs of Ebselen and cysteine's binding compounds.

The Library of Pharmacologically Active compounds (LOPAC or LOPAC^® 1280), which contains 1280 compounds representing all major target classes, was purchased from Sigma- Aldrich (St. Louis, MO). The compounds were screened at a final concentration of 10 μΜ. Ebselen was purchased from Adipogen Corporation (#AG-CR 1-0031) and Ebselen oxide from Cayman Chemical (#10012298). The 2pyr- and 3pyr-Ebselen analogs were synthesized at Wroclaw University of Technology, Poland, with purity and stability assessed at St. John's University, NY. Other Ebselen analogs, S-, O- and C-Ebselen, were provided by Dr. Barbara Slusher, Johns Hopkins University School of Medicine, Baltimore, MD. The cysteine's binding compounds, Omeprazole (Afla-Aesar #AAJ62860-03), Methyl-3,4-dephostatin (Sigma #M9440) and 3-Bromo-l,l ,l-trifluoroacetone (BTFA- Sigma #18545) were purchased.

Cloning, expression and purification of proteins.

The sequence of the C-terminal domain of NL4.3 (CTD corresponding to the last 89 amino acids of p24) was amplified by PCR and cloned in pET42b (Novagen) or pT7Flag

(Sigma-Aldrich) to generate GST-CTD and CTD-Flag constructs. CTD sequence was also cloned into pET42b deleted for GST to generate CTD-His protein (called untagged CTD), used as inhibitor of GST-CTD/CTD-Flag interaction (these constructs were provided by Dr. Massimo Caputi).

The expression and purification of the recombinant untagged CTD, GST-CTD and CTD- Flag were performed as follows, pET42b and pT7-Flag plasmids encoding CTD, GST-CTD and CTD-Flag proteins were transformed into Escherichia coli BL21(DE3) cells (Biolabs) and grown at 37°C in Luria Bertani (LB) medium until the culture reached an optical density at 600 nm of 0.6-0.8. Expression was induced by adding 1 mM IPTG and incubation was continued for 3 hrs. Cell pellets were resuspended with lysis buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA and complete EDTA-free protease inhibitor cocktail (Roche)]. After sonication, cells were centrifuged at 100,000 g for 30 min at 4°C. The soluble fraction containing CTD, GST-CTD or CTD-Flag proteins were purified by affinity chromatography on a Ni-NTA column following the manufacturer's instructions (HiTrap Ni-NTA; Amersham Biosciences). Proteins were eluted with 100, 150 and 250 mM imidazole and dialyzed overnight at 4°C against 20 mM Tris HC1 pH 8.0. The homogeneity of the purified proteins was determined by SDS-PAGE and confirmed by Western immunoblot analysis using a mouse monoclonal antibody against p24 (AIDS Reagent).

ELISA assay for monitoring GST-CTD/CTD-Flag interaction.

GST-CTD and GST proteins diluted in PBS (250 nM in 100 μΐ,) were coated onto a high- binding 96-well plate overnight at 4°C. The uncoated proteins at each step were washed out with 0.05 % Tween 20 in PBS (PBS-T). Wells were saturated with 5 % milk-PBS for 1 hr at RT and a range of concentrations of CTD-Flag protein (0 to 500 nM in 100 μΐ,) was added for 1 hr at RT in 5 % milk-PBS. Rabbit anti-Flag antibody (Rockland, cat # 600-401-384) was added at a dilution of 1 : 1000 in 5 % milk-PBS and incubated for 1 hr at RT. Then, anti-rabbit IgG-HRP antibodies (cat# 170-5047) were added at a dilution of 1 : 10000 in 0.5 % milk-PBS and incubated for 1 hr at RT. Color development was assessed using TMB super-sensitive HRP substrate (Immunochemistry Technologies). The reaction was stopped by adding TMB STOP solution (Immunochemistry Technologies) and absorbance was measured at 450 nm. Inhibition of dimerization was assessed as done previously (GST-CTD 250 nM) by adding increasing concentrations of untagged CTD (0 to 25 μΜ) to constant concentration of CTD-Flag (500 nM). CTD inhibition EC₅o was calculated via Prism software using log (inhibitor) vs response, at 3 parameters.

TR-FRET.

TR- FRET (time-resolved fluorescence resonance energy transfer) assay was used to monitor CTD domain dimerization and to screen for inhibitors of this interaction. This assay is based on the transfer of energy between two fluorophores, a donor and an acceptor labeled with europium cryptate (Eu) and allophycocyanon (XL-665), respectively (Cisbio Bioassays). The proteins, as well as the antibody-tagged fluorophores, were diluted in assay buffer [20 mM Tris- HC1 (pH 8.0), 150 mM NaCl, 10% glycerol, 0.05% Triton-XlOO and 100 mM Potassium Fluoride; pH 8.0]. In a 384 well-plate, GST-CTD and CTD-Flag (200 nM each) were mixed together with EU-conjugated anti-GST antibody (1.8 ng/well; Cisbio, cat # 61 GSTKLB) and XL-665-conjugated anti-Flag antibody (20 ng/well; Cisbio, cat # 61FG2XLB), and incubated overnight at RT. Interaction between the two proteins and illumination with a 320 nm beam resulted in transfer of fluorescence energy and emission at 665 nm. Untagged CTD was used as control of inhibition of GST-CTD/CTD-Flag dimerization.

For HTS the assay was further miniaturized to a 1536-well plate format. All concentrations were retained, yet the volume was scaled down to 5 uL. The proteins and the test compounds (10 μΜ) were mixed together and incubated for 20 min at RT then the 2 antibody-tagged fluorophores were added. The mixture was incubated for 5 hrs at RT before reading the emission at 665 nm. The TR-FRET signal was calculated as (665 nm/620 nm)* 10,000. HCV core was used as a counterscreen [60]. All plates were analyzed and passed QC if their Z' was greater than 0.5. All data was archived into the Scripps drug discovery database and activity was determined based on high and low controls. The inhibitory activity of Ebselen and its analogs was further evaluated by TR-FRET in a 384-well plate format at a 10 μΜ final concentration.

Nuclear Magnetic Resonance (NMR).

NMR data were collected on a 700 MHz Bruker NMR instrument equipped with a QCI cryoprobe. CA-CTD and WAMA were prepared in 25 mM phosphate (pH 6.5), 100 mM NaCl, 0.02% NaN₃, 10% D₂0, with or without 5 mM DTT to a final concentration of 1 mM and 330 μΜ, respectively. 2D ['H, ¹⁵N]-HSQC spectra were acquired at 298 K, with or without 1.2-fold or 2.4-fold molar excess of Ebselen prepared as 50 or 100 mM stocks in DMSO-d6. All samples contain the same amount of DMSO-d6 (2%). Chemical shifts were assigned using previously published CA-CTD [58] and WAMA HSQC peak assignments [59]. Data were processed using Bruker Topspin 3.0 and analyzed with NMRViewJ (OneMoon Scientific, Inc.).

Tryptophan Fluorescence Assay.

CA-CTD and negative control protein, lysozyme, were prepared at 5 μΜ and 2.5 μΜ respectively in the same buffer for NMR, with or without 5 mM DTT. Similarly, L-Trp was prepared at 25 μΜ, with or without DTT. Serial dilutions (1 : 1) of Ebselen and negative control ligand, Raltegravir, were prepared in DMSO and added to the protein or L-Trp samples to final concentrations ranging from 25 nM to 50 μΜ. Samples were plated in duplicate at 12 concentration points in a 96-well black quartz microplate (Hellma) and read using SpectraMax M5e at excitation and emission wavelengths of 280 nm and 335 nm, respectively. The effect of the ligand on L-Trp fluorescence is subtracted from the ligand quenching to the intrinsic Trp fluorescence of CA-CTD or lysozyme and converted to percentage with respect to the fluorescence of vehicle control samples. Data were analyzed in GraphPad Prism.

Circular Dichroism.

14 μΜ CA-CTD was buffer-exchanged in 10 mM potassium phosphate, 100 mM F, pH 6.6. For ligand-bound samples, Ebselen (in DMSO) was added at 2.4: 1 molar ratio with respect to the protein, and after one-hour incubation, was buffer exchanged anew to remove traces of DMSO. Experiments were performed in triplicate for each sample in a CD quartz cuvette (Hellma) with a path length of 1 mm. Wavelength scans from 190-250 nm at 1 nm increments were acquired at 25°C in a JASCO J-815 CD spectrometer.

Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS).

The molecular mass of CA-CTD was measured on a LTQ XL linear ion trap mass spectrometer (Thermo-Fisher Scientific) connected to a liquid chromatography (LC) system. The CA-CTD was reacted with different molar ratios of Ebselen and subsequently desalted and diluted to 4 μΜ. The resulting protein was acidified using 0.1% formic acid (FA) and separated onto a C8 column employing a 0% - 100% Acetonitrile gradient. The mass spectra were acquired in the positive ion mode. Mass-to-charge ratios were extracted from the raw data, deconvoluted and deisotoped with the MaqTran software.

Mitochondrial Metabolic Activity Assay.

Cell viability was assessed by mitochondrial metabolic activity (MTT) (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. This assay performed in HeLa- CD4, TE671 cells and PBMCs in the presence of increasing concentrations of Ebselen for 72 hrs, 48 hrs and 4 days respectively, according to the manufacturer's protocol (ATCC).

Multiple-round infection. HeLa-CD4-LTR-lacZ cells were plated at 5xl0⁴ cells per well in a 96-well plate. The next day, cells were infected with replicative NL4.3 in presence of increasing concentration of Ebselen, Efavirenz, Lopinavir or DMSO control in a total volume of 200 μί. At 72 hrs postinfection, cells were disrupted in lysis buffer (60 mM Na₂HP0₄, 40 mM NaH₂P0₄, 10 mM KC1, 10 mM MgS0₄, 2.5 mM EDTA, 50 mM β-mercaptoethanol, 0.125%Nonidet P-40) and a quantitative chlorophenol red-B-D-galactopyranoside (CPRG)-based (Boehringer Mannheim) assay was performed. The cell extracts were incubated in a reaction buffer (0.9 M phosphate buffer [pH 7.4], 9 mM MgCl₂, 1 1 mM β-mercaptoethanol, 7 mM CPRG) until a red color developed and measured at 572 nm. Experiments were performed in triplicate.

Single-round infection.

VSV-G-NL4.3 pseudotyped viruses were generated by co-transfecting 3x10⁶ 293T with 2 μg of VSV-G envelope (pMDG) and 2 μg of defective pNL4.3-eGFP using 7 <ms!T-LTl transfection reagent (Mirus). pNL4.3-eGFP is based on pNL4.3-R-E- (AIDS Reagent) and corresponds to the entire HIV-1 genome with 2 frameshifts in vpr and env, and eGFP cloned in nef using Xhol and NotI restriction sites. The culture supernatant was collected 48 hrs and 72 hrs post-transfection, filtered, aliquoted and stored at -80°C. The titer was determined in TE671 cells by Flow cytometry analysis. HeLa-CD4-LTR-lacZ were seeded at lxl 0⁴ cells per well in a 96- well plate. The next day, cells were infected with VSV-G-NL4.3-eGFP viruses in presence of increasing concentration of Ebselen, Efavirenz, Lopinavir or DMSO as control. Forty-eight hours post-infection, β-galactosidase was measured by CPRG as previously described.

Quantification of early and late reverse transcription product and provirus integration.

HeLa-CD4 cells were plated at 2x10⁵ cells per well in a 6-well plate. Twenty-four hours later, cells were infected with VSV-G-NL4.3 pseudotyped viruses in presence of Ebselen, Efavirenz, Raltegrvir, Lopinavir or DMSO. Heat-inactivated virus for 10 min at 95°C, was used as control. Eighteen hours post-infection, genomic DNAs (gDNA) were prepared using the DNeasy Blood and Tissue Kit (Qiagen), following the manufacter's protocol, and subject to quantitative PCR (qPCR) using Sensifast Sybr mix (Bioline). The early viral DNA products were amplified using the primer pair (PI) 5'-GGC TAA CTA GGG AAC CCA CTG-3' and (P2) 5'- CTG CTA GAG ATT TTC CAC ACT GAC-3', and the late viral DNA products were amplified using the primer pair (P3) 5'-TGT GTG CCC GTC TGT TGT G-3' and (P4) 5'-GAG TCC TGC GTC GAG G-3 ' [16]. The number of integrated pro viruses was quantified by Alu-Gag PCR followed by a nested qPCR [83]. The Alu-PCR was run with the following conditions: initial denaturation of 2 min at 94°C, then 20 cycles of amplification of 20 s at 94°C, 10 s at 50°C, and 3.5 min at 65°C, and a final elongation step of 7 min at 65°C. Primers sequences were as follow: (Alu) 5' -TCC CAG CTA CTG GGG AGG CTG AGG-3'; (gag) 5'-CCT GTG TCA GCT GCT TG-3'; nested q-PCR done with primers PI and P2. The qPCR amplification was run as follow: 3 min at 95°C, then 40 cycles of 5 s at 95°C, 20 s at 60°C, and 8 s at 68°C.

Isolation and infection of PBMCs.

PBMCs were isolated from buffy coat as previously described [72]. Briefly, 25 mL of total blood diluted 2 fold in PBS were carefully laid over 14 mL of Ficoll-paque and centrifuged at 2000 rpm for 25 min at 20°C (brakes off). PBMCs ring cell was carefully removed and transferred to a new 50 mL tube. Enough PBS was added to the PBMCs to fill up to 50 mL and centrifuged at 1500 rpm for 10 min at 20°C (brakes on). The cell pellet was washed twice with PBS and finally recovered in 30 mL of RPMI 1640 supplemented with 10% serum, 1%

Penicillin-streptomycin. PBMCs were counted and plated at lxl 0⁶ cells/mL. To activate the PBMCs, PHA (phytohaemagglutinin) was added at 3 μg/mL. Two to three days later 20 U/mL of Interleukin 2 (IL-2) were added.

PHA activated PBMCs were centrifuged and recovered at lxlO⁶ cells per well in a 6-well plate, and infected with NL4.3 in presence of increasing concentration of Ebselen for 6 hrs. Cells were washed 3 times in PBS and resuspended in 2 mL complete RPMI (+ 20 U/mL IL2) containing drugs. Four days post infection, the supernatant was recovered for p24 ELISA assay.

Fate of CA in cells.

HeLa-CD4 cells were plated at 2xl0⁶ cells in a 10 cm plate. Twenty-four hours later, cells were infected with NL4.3-eGFP virus for 30 min at 4°C to allow viral attachment to cells, and then were moved to 37°C for 16 hrs in presence of 20 μΜ Ebselen, 6 μΜ PF74 or DMSO. Cells were washed 3 times with ice-cold PBS, and then detached with 1 mL of pronase (7.0 mg/mL prepared in DMEM) for 5 min at room temperature. Cell pellets were washed 3 times in PBS and resuspended in 2.5 mL of hypotonic buffer [10 mM tris-HCl pH8; 10 mM KC1; 1 mM EDTA and protease inhibitors] for 15 min in ice. Cells were lyzed with 15 strokes in a 7 mL Dounce homogenizer with pestle B and cellular debris were cleared by centrifugation for 3 min at 2000 g. Cleared lysate (100 μί,) was harvested and used as input for HIV-1 p24. Two mL of cleared lysate were ultracentrifuged onto a 50% sucrose cushion (weight:volume, in PBS) at 125,000 g for 2 hrs at 4°C (SW41 Beckman rotor). The top-most part (100 μί) of the supernatant containing soluble CA was harvested. The pellet, containing fullerene CA, was resuspended in 100 \xL SDS sample buffer. The input, supernatant and pellet fractions were run on SDS-PAGE followed by western-blot with anti-p24 antibody.

In vitro CA assembly assay.

The effect of Ebselen on CA assembly was measured by monitoring the turbidity at 350 nm as previously described [38]. Briefly, 75 μΐ of NaCl solution (2 mL of 5M NaCl mixed with 1 mL of 200 mM sodium phosphate, pH8) containing increasing concentration of Ebselen, PF-74, Raltegravir (used as negative control) or DMSO were placed into a 96-well plate. To initiate the assembly reaction, 25 μΐ of purified P24-His protein (100 μΜ; 25 μΜ final) was added. Turbidity was monitored every 30 s for 20 m.

Impact of Ebselen on MoMuLV infection.

VSV-G-MoMuLV-pseudotyped viruses were generated by co-transfecting 5xl 0⁶293T in 15 cm² plates with a combination of 2 μg of VSV-G envelope (pMDG), 2 μg of MoMuLV Gag- Pol plasmid (pHit60) and 4 μg of pCNCG (MLV genome encoding a CMV-driven eGFP) using J ms!T-LTl transfection reagent (Mirus). The culture supernatant was collected at 48 hrs and 72 hrs post-transfection, filtered, aliquoted and stored at -80°C. The titer was determined in TE671 cells by Flow cytometer analysis. Infectivity assays were assessed in TE671 plated at 7.5x10⁴ cells per well in a 12-well plate. The day after, cells were infected with VSV-G- MoMuLV-pseudotyped viruses in complete media containing polybrene (5 μg/mL), in the presence of increasing concentrations of Ebselen. Two days later, cells were washed, pelleted and then GFP-positive cells were analyzed using a flow cytometer (Accuri Sampler).

Impact of Ebselen on SIV infection. CEM174 cells were infected with SIVmac239 for 6 hours, then washed and plated at 5xl 0⁵ cells per well in a 6- well plate. Cells were treated with increasing concentrations of Ebselen or DMSO for 72 hours. The impact of Ebselen on SIV replication was assessed by p27 ELISA on culture supernatant.

Impact of Ebselen on HCV infection.

The impact of Ebselen on HCV infection was evaluated by replication assay and infectivity assay. The replication assay was monitored in Huh-7.5 cells containing pSG-Rluc-2a- neo-NS3-5B JFHl (subtype 2a). 2.5x10⁵ PSG-Rluc-2a-neo-JFHl replicon cells were seeded per well in a 24-well plate. The next day, cells were treated with Ebselen (5 and 10 μΜ), Raltegravir (1 μ ), BMS-052 Active compound (10 nM), or DMSO control. The BMS-790052 Active compound targets the HCV NS5A protein, and disrupts both viral replication and assembly. Seventy-two hrs later, cells were washed in PBS, lysed in IX Renilla luciferase lysis buffer and relative light units (RLU) was measured using the Renilla luciferase assay (Promega) on a Berthold luminometer. Luciferase signals from this replicon cells system directly correlate with HCV RNA replicon levels. Experiments were performed in triplicate. Infectivity assay was performed as follow: 1 .25x10⁵ Huh-7.5 cells were seeded per well in a 24-well plate. Twenty- four hrs later, cells were treated with Ebselen (5 and 10 μΜ), Raltegravir (1 μΜ), BMS-052 Active compound (10 nM), or DMSO control. The day after, cells were treated with a cell- culture adapted strain of JC 1 ( JC 1.1 ; subtype 2a) at an MOI of 0.5 for 5 hrs, then culture supernatant was replaced by fresh media. Forty-eight hrs post-infection, the viral supernantant were harvested, serially diluted and subsequently used to infect naive Huh-7.5 cells seeded 24 hrs before in a 96-well plate coated with poly-l-lysine at a density of 8x l 0⁴ cells per well. Forty- eight hrs later, titers were analyzed using immunohistochemistry to detect infected cells by 9E10 anti-NS5A antibody (Provided by Dr. Charles Rice of Rockefeller University) staining as described [84]. The unbounded antibodies at each step were washed out 2X with PBS and IX with PBS + 0.1 % saponin. Cells were fixed with 200 iL cold methanol for 10 min at room temperature, washed 2X with PBS and incubated in 50 μΕ/well Blocking Buffer (BSA 1%, skim milk 0.2% in IX PBS, and 1 % saponin) for lhr at RT. Blocking buffer was removed and endogenous peroxidase was blocked via incubation with PBS + 0.3% hydrogen peroxide (H₂0₂) for 5 min at RT. Cells were washed and then incubated for 1 hr at RT in 50 μΐ/ννβΐΐ of purified anti-NS5A 9E10 monoclonal at a 1 : 10000 dilution of a 1 mg/mL stock in PBS + 0.1% saponin. Using the ImmPRESS Reagent Kit (Vector Labs), an anti-mouse Ig (Cat# MP-7402) at a 1 :3 dilution in PBS + 0.1% saponin was added for lhr at RT in 50 μίΛνβΙΙ. The reaction was developed using DAB chromatogen substrate (Vector Labs) for 5-20 min at RT in 50 μίΛνεΙΙ according to the manufacturer's instructions. The reaction was stopped by washing 2X with PBS and positive wells were counted via microscope. 50% Tissue Culture Infectious Dose/mL (TCID₅₀/mL) was determined using the Reed and Muench calculator as described previously [84].

Impact of Ebselen on influenza infection.

Influenza A virus (H1N1 , A / Virginia / ATCC1 / 2009, ATCC VR-1736) produced in MDCK cells cultured in serum-free OptiPRO medium (Life Technologies), was mixed with Ebselen (1 and 10 μΜ), Raltegravir (1 μΜ), Aleuria Aurantia Lectin (100 and 300 nM) or DMSO control, and used to infect Hela-CD4 cells at 37°C. After one hr the culture supematants were removed and cells were further grown in fresh DMEM supplemented with 10% FBS until the next day. Infected cells were then trypsinized, fixed with 1% paraformaldehyde in PBS, permeabilized in 0.1% saponin in PBS containing 2% goat serum and stained with a mouse monoclonal antibody recognizing H1N1/H2N2 (clone C179, Takara), followed by an Alexa647- conjugated goat anti-mouse antibody (Jackson Immunoresearch). Cells were then washed, fixed in 2% paraformaldehyde in PBS, and analyzed by flow cytometry (BD Biosciences C6 Accuri cytometer).

CITED DOCUMENTS

1. Ambrose, Z. and C. Aiken, HIV-1 uncoating: connection to nuclear entry and regulation by host proteins. Virology, 2014. 454-455: p. 371-9.

2. Arhel, N., Revisiting HIV-1 uncoating. Retrovirology, 2010. 7: p. 96.

3. Ganser-Pornillos, B.K., M. Yeager, and 0. Pornillos, Assembly and architecture of HIV.

Adv Exp Med Biol, 2012. 726: p. 441-65.

4. Sundquist, W.I. and H.G. Krausslich, HIV-1 assembly, budding, and maturation. Cold Spring Harb Perspect Med, 2012. 2(7): p. a006924.

5. Hulme, A.E., 0. Perez, and T.J. Hope, Complementary assays reveal a relationship between HIV-1 uncoating and reverse transcription. Proc Natl Acad Sci U S A, 2011. 108(24): p. 9975-80.

6. Forshey, B.M., et al., Formation of a human immunodeficiency virus type 1 core of optimal stability is crucial for viral replication. J Virol, 2002. 76(11): p. 5667-77. Wacharapornin, P., D. Lauhakirti, and P. Auewarakul, The effect of capsid mutations on HIV-1 uncoating. Virology, 2007. 358(1): p. 48-54.

Yufenyuy, E.L. and C. Aiken, The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid. Retrovirology, 2013. 10: p. 29. Hulme, A.E., et al., Identification of capsid mutations that alter the rate of HIV-1 uncoating in infected cells. J Virol, 2015. 89(1): p. 643-51.

Black, L.R. and C. Aiken, TRIM5alpha disrupts the structure of assembled HIV-1 capsid complexes in vitro. J Virol, 2010. 84(13): p. 6564-9.

Perron, M.J., et al., The human TRIM5alpha restriction factor mediates accelerated uncoating of the N-tropic murine leukemia virus capsid. J Virol, 2007. 81(5): p. 2138- 48.

Stremlau, M., et al., The cytoplasmic body component TRIM5aIpha restricts HIV-1 infection in Old World monkeys. Nature, 2004. 427(6977): p. 848-53.

Stremlau, M., et al., Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc Natl Acad Sci U S A, 2006. 103(14): p. 5514-9.

Fricke, T., et al., MxB binds to the HIV-1 core and prevents the uncoating process of HIV-1. Retrovirology, 2014. 11: p. 68.

Goujon, C, et al., Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection. Nature, 2013. 502(7472): p. 559-62.

Liu, Z., et al., The interferon-inducible MxB protein inhibits HIV-1 infection. Cell Host Microbe, 2013. 14(4): p. 398-410.

Abdurahman, S., et al., Selected amino acid substitutions in the C-terminal region of human immunodeficiency virus type 1 capsid protein affect virus assembly and release. J Gen Virol, 2004. 85(Pt 10): p. 2903-13.

Abdurahman, S., et al., Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity. Retrovirology, 2007. 4: p. 69.

Chang, Y.F., et al., Mutations in capsid major homology region affect assembly and membrane affinity of HIV-1 Gag. J Mol Biol, 2007. 370(3): p. 585-97.

Douglas, C.C., et al., Investigation of N-terminal domain charged residues on the assembly and stability of HIV-1 CA. Biochemistry, 2004. 43(32): p. 10435-41.

Ganser-Pornillos, B.K., et al., Assembly properties of the human immunodeficiency virus type 1 CA protein. J Virol, 2004. 78(5): p. 2545-52.

Joshi, A., K. Nagashima, and E.O. Freed, Mutation of dileucine-like motifs in the human immunodeficiency virus type 1 capsid disrupts virus assembly, gag-gag interactions, gag-membrane binding, and virion maturation. J Virol, 2006. 80(16): p. 7939-51. Lopez, C.S., et al., Determinants of the HIV-1 core assembly pathway. Virology, 2011. 417(1): p. 137-46.

Deshmukh, L., et al., Structure and dynamics of full-length HIV-1 capsid protein in solution. J Am Chem Soc, 2013. 135(43): p. 16133-47.

Pornillos, 0., et al., X-ray structures of the hexameric building block of the HIV capsid. Cell, 2009. 137(7): p. 1282-92.

Pornillos, 0., B.K. Ganser-Pornillos, and M. Yeager, Atomic-level modelling of the HIV capsid. Nature, 2011. 469(7330): p. 424-7. Shin, R., Y.M. Tzou, and N.R. Krishna, Structure of a monomeric mutant of the HIV-1 capsid protein. Biochemistry, 2011. 50(44]: p. 9457-67.

Zhao, G., et al., Mature HIV-1 capsid structure by cryo-electron microscopy and all- atom molecular dynamics. Nature, 2013. 497(7451): p. 643-6.

Gitti, R.K., et al., Structure of the amino-terminal core domain of the HIV-1 capsid protein. Science, 1996. 273(5272}: p. 231-5.

Byeon, I.J., et al., Structural convergence between Cryo-EM and NMR reveals intersubunit interactions critical for HIV-1 capsid function. Cell, 2009. 139(4): p. 780- 90.

Gamble, T.R., et al., Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science, 1997. 278(5339): p. 849-53.

Worthylake, D.K., et al., Structures of the HIV-1 capsid protein dimerization domain at 2.6 A resolution. Acta Crystallogr D Biol Crystallogr, 1999. 55(Pt 1): p. 85-92.

Lanman, J., et al., Kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type 1 capsid protein assembly in vitro. J Virol, 2002. 76(14): p. 6900-8.

von Schwedler, U.K., et al., Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J Virol, 2003. 77(9): p. 5439-50.

Ganser-Pornillos, B.K., A. Cheng, and M. Yeager, Structure of full-length HIV-1 CA: a model for the mature capsid lattice. Cell, 2007. 131(1): p. 70-9.

Schur, F.K., et al., Structure of the immature HIV-1 capsid in intact virus particles at 8.8 A resolution. Nature, 2015. 517(7535): p. 505-8.

Thenin-Houssier, S. and S.T. Valente, HIV-1 Capsid Inhibitors as Antiretroviral Agents. Curr HIV Res, 2016. 14(3): p. 270-82.

Kortagere, S., et al., Inhibiting early-stage events in HIV-1 replication by small- molecule targeting of the HIV-1 capsid. J Virol, 2012. 86(16): p. 8472-81.

Tang, C, et al., Antiviral inhibition of the HIV-1 capsid protein. J Mol Biol, 2003. 327(5): p. 1013-20.

Curreli, F., et al., Virtual screening based identification of novel small-molecule inhibitors targeted to the HIV-1 capsid. Bioorg Med Chem, 2011. 19(1): p. 77-90. Goudreau, N., et al., Novel inhibitor binding site discovery on HIV-1 capsid N-terminal domain by NMR and X-ray crystallography. ACS Chem Biol, 2013. 8(5): p. 1074-82. Lemke, C.T., et al., Distinct effects of two HIV-1 capsid assembly inhibitor families that bind the same site within the N-terminal domain of the viral CA protein. J Virol, 2012. 86(12): p. 6643-55.

Fader, L.D., et al., Discovery of a l,5-dihydrobenzo[b][l,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly. Bioorg Med Chem Lett, 2011. 21(1): p. 398-404. Lamorte, L, et al., Discovery of novel small-molecule HIV-1 replication inhibitors that stabilize capsid complexes. Antimicrob Agents Chemother, 2013. 57(10): p. 4622-31. Cao, J., et al., High-throughput human immunodeficiency virus type 1 (HIV-1) full replication assay that includes HIV-1 Vif as an antiviral target. Antimicrob Agents Chemother, 2005. 49(9): p. 3833-41.

Sticht, J., et al, A peptide inhibitor of HIV-1 assembly in vitro. Nat Struct Mol Biol, 2005. 12(8): p. 671-7. Garzon, M.T., et al., The dime zation domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: a biophysical characterization. Protein Sci, 2004. 13(6): p. 1512-23.

Lampel, A., et al., Targeting the Early Step of Building Block Organization in Viral Capsid Assembly. ACS Chem Biol, 2015.

Blair, W.S., et al., HIV capsid is a tractable target for small molecule therapeutic intervention. PLoS Pathog, 2010.6(12): p. el001220.

Lemke, C.T., et al., A novel inhibitor-binding site on the HIV-1 capsid N-terminal domain leads to improved crystallization via compound-mediated dimehzation. Acta Crystallogr D Biol Crystallogr, 2013.69(Pt 6): p. 1115-23.

Zhang, H., et al., A cell-penetrating helical peptide as a potential HIV-1 inhibitor. J Mol Biol, 2008.378(3): p. 565-80.

Zhang, H., et al., Antiviral activity of alpha-helical stapled peptides designed from the HIV-1 capsid dimehzation domain. Retrovirology, 2011.8: p. 28.

Zhang, H., et al., Dual-acting stapled peptides target both HIV-1 entry and assembly. Retrovirology, 2013.10: p. 136.

Martin, D.E., et al., Safety and pharmacokinetics of Bevirimat (PA-457), a novel inhibitor of human immunodeficiency virus maturation, in healthy volunteers. Antimicrob Agents Chemother, 2007.51(9): p. 3063-6.

Smith, P.F., et al., Phase I and II study of the safety, virologic effect, and pharmacokinetics/pharmacodynamics of single-dose 3-o-(3',3'- dimethylsuccinyljbetulinic acid (bevirimat) against human immunodeficiency virus infection. Antimicrob Agents Chemother, 2007.51(10): p. 3574-81.

Adamson, C.S., et al., Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1 maturation inhibitor bevirimat. Retrovirology, 2010.7: p. 36. Van Baelen, K., et al., Susceptibility of human immunodeficiency virus type 1 to the maturation inhibitor bevirimat is modulated by baseline polymorphisms in Gag spacer peptide 1. Antimicrob Agents Chemother, 2009.53(5): p. 2185-8.

Jung, J., et al., 1H, 15N and 13C assignments of the dimehc C-terminal domain of HIV-1 capsid protein. Biomol NMR Assign, 2010.4(1): p. 21-3.

Wong, H.C., R. Shin, and N.R. Krishna, Solution structure of a double mutant of the carboxy -terminal dimehzation domain of the HIV-1 capsid protein. Biochemistry, 2008.47(8): p. 2289-97.

Kota, S., et al., A time-resolved fluorescence-resonance energy transfer assay for identifying inhibitors of hepatitis C virus core dimehzation. Assay Drug Dev Technol, 2010.8(1): p. 96-105.

Azad, G.K. and R.S. Tomar, Ebselen, a promising antioxidant drug: mechanisms of action and targets of biological pathways. Mol Biol Rep, 2014.41(8): p. 4865-79. Li, M., et al., First-in-class small molecule inhibitors of the single-strand DNA cytosine deaminase APOBEC3G. ACS Chem Biol, 2012.7(3): p. 506-17.

Sakurai, T., et al., Ebselen, a seleno-organic antioxidant, as an electrophile. Chem Res Toxicol, 2006.19(9): p. 1196-204.

Favrot, L., D.H. Lajiness, and D.R. Ronning, Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors. J Biol Chem, 2014. 289(36): p. 25031-40. 65. McDermott, J., et al., Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents. J Virol, 1996. 70(8): p. 5106-14.

66. Lambrecht, N., et al., Identification of the site of inhibition by omeprazole of a alpha- beta fusion protein of the Η,Κ-ATPase using site-directed mutagenesis. } Biol Chem, 1998. 273(22): p. 13719-28.

67. Didenko, T., et al., Fluorine-19 NMR of integral membrane proteins illustrated with studies ofGPCRs. Curr Opin Struct Biol, 2013. 23(5): p. 740-7.

68. Ternois, F., et al., The HIV-1 capsid protein C-terminal domain in complex with a virus assembly inhibitor. Nat Struct Mol Biol, 2005. 12(8): p. 678-82.

69. Yang, Y., J. Luban, and F. Diaz-Griffero, The fate of HIV-1 capsid: a biochemical assay for HIV-1 uncoating. Methods Mol Biol, 2014. 1087: p. 29-36.

70. Bhattacharya, A., et al., Structural basis of HIV-1 capsid recognition by PF74 and CPSF6. Proc Natl Acad Sci U S A, 2014. 111(52): p. 18625-30.

71. Barklis, E., et al., Characterization of the in vitro HIV-1 capsid assembly pathway. J Mol Biol, 2009. 387(2): p. 376-89.

72. Mousseau, G., et al., An analog of the natural steroidal alkaloid cortistatin A potently suppresses Tat-dependent HIV transcription. Cell Host Microbe, 2012. 12(1): p. 97- 108.

73. Thomas, A.G., et al., Kinetic characterization of ebselen, chelerythrine and apomorphine as glutaminase inhibitors. Biochem Biophys Res Commun, 2013. 438(2): p. 243-8.

74. Pino, M.A., M. Pietka-Ottlik, and B. Billack, Ebselen analogues reduce 2-chloroethyl ethyl sulphide toxicity in A-431 cells. Archives of Industrial Hygiene and Toxicology, 2013. 64(1): p. 77-86.

75. von Schwedler, U.K., et al., Proteolytic refolding of the HIV-1 capsid protein amino- terminus facilitates viral core assembly. EMBO J, 1998. 17(6): p. 1555-68.

76. Cheslock, S.R., et al., Charged assembly helix motif in murine leukemia virus capsid: an important region for virus assembly and particle size determination. J Virol, 2003. 77(12): p. 7058-66.

77. Wang, M.Q. and S.P. Goff, Defects in virion production caused by mutations affecting the C-terminal portion of the Moloney murine leukemia virus capsid protein. J Virol, 2003. 77(5): p. 3339-44.

78. Mukherjee, S., et al., Ebselen inhibits hepatitis C virus NS3 helicase binding to nucleic acid and prevents viral replication. ACS Chem Biol, 2014. 9(10): p. 2393-403.

79. Jubb, H., et al., Structural biology and drug discovery for protein-protein interactions.

Trends Pharmacol Sci, 2012. 33(5): p. 241-8.

80. Li, G., et al., Functional conservation of HIV-1 Gag: implications for rational drug design. Retrovirology, 2013. 10: p. 126.

81. Kil J, L.E., Griffiths S, Lobarinas E, Spankovich C, Antonelli PJ, Le Prell C, Efficacy of SPI-1005 for prevention of Noise-Induced Hearing Loss: Phase 2 Clinical Trial Results. Otolaryngol Head Neck Surg, 2014. 151 no. 1 suppl(no. 1 suppl): p. P83-P84.

82. Bauer, R.A., Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies. Drug Discov Today, 2015. 20(9): p. 1061- 73.

83. Butler, S.L., M.S. Hansen, and F.D. Bushman, A quantitative assay for HIV DNA integration in vivo. Nat Med, 2001. 7(5): p. 631-4. 84. Lindenbach, B.D., et al., Complete replication of hepatitis C virus in cell culture. Science, 2005. 309(5734): p. 623-6.

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements will be apparent to those skilled in the art without departing from the spirit and scope of the claims.

All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

What is claimed is:

1 , A method for the inhibition of assembly and disassembly of HIV- 1 capsid, eomprisinj contacting a cell or a population of cells infected with HI^'V-l and an effective amount or concentration of a compound of formula !)

wherein

ring A is a 5- or 6-membered aryl or heteroaryi ring fused to the ring comprising atom X; ring B is a 5- or 6-membered ary! or heieroaryl, wherein atom Z is bonded to a carbon atom thereof;

Q is C(=0) or S0₂;

X is Se, Se(O), S, S(O), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (C3 -C4)alkyl);

J is independently at each occurrence (C 1 -C4)aikyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (Cl-C4)alkoxy_i (Cl-C4)haloalkoxy_s halo, cyano, nitro, (Cl-C4)alkyl-S(0)_q wherein q = 0, 1, or 2, (C3-C4)alkyl-N(R)S0₂, (CI-C4)aIkyl-8G₂M(R), (C1 -C4)alkyl- N(R)C(-0)_s (Cl-C4)alkyl-C(=0)N(R), (Cl -C4)alkyl-OC(=0), (CI~C4)alkyl-C( ))0, (Cl - C4)alkyl-OC(-0)N(R)_s or (Cl-C4)aJkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5~ to 7-membered heteroaryi, or 6- to 10-membered aryl, any of which can be unsubstit ted or substituted;

nl ^::· 0, L 2, 3, or 4; n2 ^:::: 0, 1, 2, 3, or 4; or a pharmaceutically acceptable salt thereof,

2, The method of claim 1, wherein the compound is a compound of formula (IA)

wherein

X is Se, Se(0), S, S(O) or S(0)₂; wherein the ring directly bonded to group X can further comprise one or two nitrogen atoms therewithin;

Y is CR or ;

Z is or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-€4)alkyL (C3-C7)cycloalkyl, (Ci- C4)haloalkyI, (Cl-C4)alkoxy_s (Cl-C4)lialoa!koxy, balo, cyano, nitro, (Cl-C4)alkyl~S(0)_q wherein q - 0, l , or 2₅ (Cl-C4)alkyl-N(R)S0_2j (Cl-C4)slkyl^»S0₂N{R)₅ (Cl-C4)alkyl- N(R)C(=0), (Cl-C )alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)i0k l-C(=O)O, (Cl-

C4)8lkyl-OC(=0)N(R)_s or (Cl~C4)alkyI~N(R)C(=0)0_! or is a 5~ to 7-membered heterocyclyl, 5- to 7-membered heteroaryi, or 6- to 10-membered ary l, any of w ch can be unsubstituied or substituted;

nl ^::: 0, 1 , 2, 3, or 4; n2 ^::: 0, 1, 2, 3, or 4;

or a pharmaceutically acceptable salt thereof,

3, The method of claim 1, wherein the compoimd of fomiuia (I) is any one of the following formulas;

or a pharmaceutically acceptable salt thereof.

4, A method of treatment or inhibition of an HIY-1 infection in a human, comprising administering to a patient afflicted therewith an effective dose of a com ound of formula (I)

wherein

ring A is a 5- or 6~membered aryl or hsteroaryi ring fused to the ring comprising atom X; ring B is a 5 - or 6-raembered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;

Q is C(=0) or S0₂;

X is Se, Se(0)_? S_s 8(G), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyL (Cl~

C4)haioalkyl, (Cl-C4)alkoxy, (Cl-C4)haloalkoxy₅ halo, cyano, nitro, (Cl-C4)alkyl-S(0)_q wherein q = 0, 1 , or 2₅ (Cl-C4}a!kyl-N(R)SO_¾ (Cl-C4)alkyl-S0₂N(R)₅ (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alk l-OC(=0), (Cl-C4)alk l-C(=0)0, (CI- C4)alkyi-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(= ))0, or is a 5- to 7-rnembered heterocyclyL 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be unsubstituted or substituted;

nl 0_} \, 2, 3, or 4; n2 ···^' 0₅ 1. 2, 3, or 4; or a pharmaceutically acceptable salt thereof;

5. The method of claim 4, wherein the compound is a compound of formula (I A)

wherein

X is Se, Se(G), S, S(0) or S(0)₂:

wherein the ring directly bonded to group X can further comprise one or two nitrogen atoms therewithal; Y is CR or N;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl_s (C3-C7)cycloalkyl, (Cl- C4)haIoalkyl, (Cl-C4)alkoxy₅ (Cl-C4)haloalkoxy, halo, cyaiio, nitro, (Cl -C4)alky!-S(0)_(! wherein q - 0, 1, or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl~C4)alkyl~S0₂N(R)₅ (Cl-C4)alkyl- N{R)C(-0}₅ (Cl-C4)alkyI-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl-OC(-0)N(R), or (Cl-C4)alkyl-N( )C(=0)0, or is a 5- to 7-membered heterocyclyl, 5· to 7-membered heteroaryi, or 6~ to lO-membered aryl, any of which can be ^substituted or substituted;

nl ^{; ;} 0, 1, 2, 3, or 4; n2 ^::: 0, 1, 2, 3, or 4;

or a pharmaceutically acceptable salt thereof

6, The method of claim 4, wherein the compound of formula (I) is any one of the following formulas;

or a pharmaceutically acceptable salt thereof.

7. Use of a compound of formula 1)

wherein

ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X; ring B Is a 5- or 6-membered aryl or heteroaryl wherein atom Z is bonded to a carbon atom thereof;

Q is C(=0) or S0₂;

X is Se, Se(O), S₅ S(O), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloaikyl, (Cl- C4)haloalky!_s (Cl~C4)alkoxy, (Cl-C4)haioalkoxy, halo, cyano, Hiiro, (Cl-C4)alkyl-S(0)_q wherein q = 0. 1, or 2, (Cl-C4)alkyl-N(R)SO¾ (Cl-C4)aikyl-S0₂N(¾)_S (C 1 -C4)alkyl- N(R)C(=0), (Cl-C4)aikyl-C(-0)N( }₅ (C I -C4}aikyl-OC{^::::0), (Cl-C4)alkyl-C(=0)0, (C!- C4)alkyl-GC{=0)N(R), or (C!-C4)aSkyl-N{R)C(==0)G, or is a 5~ to 7-membered heterocyclyi 5 - to 7-membered heteroaryl, or 6» to 10~mambered aryl, any of which can be w substituted or substituted;

nl - 0, 1, 2, 3, or 4; n2 ^:;; 0, 1, 2, 3_» or 4; or a pharmaceutically acceptable salt thereof; for treatment or inhibition of an HIV-1 infection in a human patient,

8- The use of claim 7, wherein the compound is of formula (I A)

wherein

X is Se₅ Se{0)₃ S, S(0) or S(0)₂;

Y is CR or N;

Z is N or CR; R is independently at each occurrence H or (Cl-C4)aikyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloa!kyi_s {€!- C4)haloalkyi_s (Cl-C4)alkoxy, (Cl~C4)haioal.koxy, halo, cyano, nitre, (Cl-C4)alkyl-S(0)_q wherein q = 0, 1, or 2, (Cl-C4)alk l-N(R)S02, (Cl-C4)alkyi-S0₂N( ), (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyI-OC(=0)N(R)_} or (C] -C4)alky!-N(R)C(=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6~ to 10-membered aryl, any of which can be imsubsticuled or substituted:

nl^■= 0j 1 , 2, 3, or 4: r¾2 ^;:: 0, 1, 2, 3, or 4;

or a pharmaceutically acceptable salt thereof,

9, The use of claim 7, wherein the compound of formula (I) is any one of the following formulas

or a pharmaceutically acceptable salt thereof.

10, Use of a compound of formula (I)

wherein

ring A is a 5- or 6~raeinbered aryl or heteroaryl ring fused to the ring comprising atom X; ring B is a 5- or 6-membeied aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;

Q is C(=0) or S0₂;

X is Se, Se(O), S_? S(O), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)aikyl)

J is independently at each occurrence (C! -€4)a!kyJ_s (C3-C7)cycloalkyl, (CI- C4)haloalkyl, (Cl-C4)alkoxy, (Cl-C4)haloaikoxy, halo, cyano, nitro, (Cl-C4)aikyl-S(0)_q wherein q = 0, 1, or 2_} (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-S0₂N(R)_S (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0)_} (Cl -C4)alkyl-C(=0)0, (CI- C4)aIkyl-OC(=0)N(R), or (Cl-C4)alkyl-N(R)C(-^:0)0₃ or is a 5- to 7-membered heterocyclyl. 5- to 7-membered heteroaryl, or 6- to IG-menibered aryl any of which can be unsubstituted or substituted;

nl = 0, L 2, 3₅ or 4; ri2. 0, 1, 2, 3, or 4; or a pharmaceutically acceptable salt thereof; for preparation of a medicament for treatment or inhibition of an HIV-1 infection in a human patient.

11 , The use of claim 10, wherein the compound is of formula (I A.)

wher

X is S , Se(O), S, S(O) or S(0)₂;

wherein the ring directly bonded to group X can further comprise one or two nitrogen atoms therewithm;

Y is CR or N;

Z is N or CR; R is independently at each occurrence H or (Cl-€4)alkyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (C I - C4)haloaIkyl, (Cl-C4)alkoxy_s (Cl-C4)haloaJkoxy_s halo, cyano, nitro, (Cl -C4)a!kyl-S(0)_q wherein q - 0_S I , or 2, (Cl-C4)alkyl-N(R)S0₂, (Cl-C4)alkyl-.SO_aN( )_} (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N( ), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl- C4)alkyl~OC(^0)N(R)_> or (Cl-C4)alkyI-N( )C(=0)0, or is a 5- to 7-membered hcterocyclyl, 5 to 7-membered heteroaryl, or 6~ to 10-membered axyl, any of which can be unsubstituted or substituted;

nl = 0, 1 , 2, 3, or 4; n2 ===- 0, L 2, 3. or 4;

or a pharmaceutically acceptable salt thereof,

12, The use of claim 10, wherein the compound of formiila (!) is any one of the following formulas

or a pharmaceutically acceptable salt thereof.

13. A compound of formula (I)

wherein

ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;

Q is C(=0) or SQ₂;

X is Se, Se(0), S, S(0), or S(0)₂;

Z is N or CR;

R is independently at each occurrence H or (Cl-C4)a_kyl);

J is independently at each occurrence (Cl-C4)alkyl, (C3-C7)cycloalkyl, (Cl- C4)haloalkyl, (Cl-C4)alkoxy, (Cl-C4)haloa1koxy₅ halo, cyano, nitro, (Cl-C4)a3kyi-S(G)_q wherein q = 0, 1, or 2, (Cl-C4)alkyl-N(R)SOa, (Cl-C4) lkyi-S02^'N(R)₅ (Cl-C4)alkyl- N(R)C(-0}_} (Cl-C4)alkyl-C(=0)N( ), (Cl~C4)aIkyl~0C(O)_s {Cl-C4)alkyI~C(=0)0, (CI- C4)aikyl-OC(=0)N(R)₉ or (Cl-C4)alkyl-N(R)C(=0)0, or is a 5- to 7-membered heterocyclvl, to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be imsubstituted or substituted;

nl ^~ 0, 1, 2, 3, or 4; n2 ^::: 0, 1, % 3. or 4; or a pharmaceutically acceptable salt thereof; wherein the compound is not ebselen.

The compound of claim 13, wherein the compound is a compound of formula (I A)

w.

X is Se_s Se(0), 8, S(0) or S(0)₂;

wherein the ring directly bonded to group X can further comprise one or two nitrogen atoms therewilhin;

Y is CR or N;

Z is N or CR;

is independently at each occurrence H or (Cl-C4)alkyl); J is independently at each occurrence (Cl-C4)a!kyl., (C3-C7)cycloalkyl_s (Cl- C4)ha!oalkyI, (Cl-C4)alkoxy. (C1 -C4)haloalkoxy, halo, cyano, niiro, (Cl~C4)alkyi~S(0)_q wherein q - 0, 1, or 2, (Cl"C4)aikyl-N(K)SQ₂, (Cl-C4)alkyl-S0₂N(R)₅ (Cl-C4)alkyl- N(R)C(=0), (Cl-C4)alkyl-C(=0)N(R), (Cl-C4)alkyl-OC(=0), (Cl-C4)alkyl-C(=0)0, (Cl-

C4)alkyl-0C(=O)N(R), or (CI -C4)a!kyl-N(R)C(^s=0)0, or is a 5- to 7-membered heterocyclyl, 5- to 7-membered heteroaryl, or 6- to 10-membered aryl, any of which can be mi substituted or substituted;

nl - 0, 1, 2, 3, or 4; a2 ^::: 0, 1, 2, 3, or 4:

or a pharmaceutically acceptable salt thereof.

15 , The compound of claim 13 of an of the following formulas:

or a pharmaceutically acceptable salt thereof.