Discovery of dihydroxyindole-2-carboxylic acid derivatives as dual allosteric HIV-1 Integrase and Reverse Transcriptase associated Ribonuclease H inhibitors

Journal Pre-proof Discovery of dihydroxyindole-2-carboxylic acid derivatives as dual allosteric HIV-1 Integrase and Reverse Transcriptase associated Ribonuclease H inhibitors Francesca Esposito, Mario Sechi, Nicolino Pala, Adele Sanna, Pratibha Chowdary Koneru, Mamuka Kvaratskhelia, Lieve Naesens, Angela Corona, Nicole Grandi, Roberto di Santo, Vincenzo Maria D'Amore, Francesco Saverio Di Leva, Ettore Novellino, Sandro Cosconati, Enzo Tramontano PII:

S0166-3542(19)30555-8

DOI:

https://doi.org/10.1016/j.antiviral.2019.104671

Reference:

AVR 104671

To appear in:

Antiviral Research

Received Date: 27 September 2019 Revised Date:

29 November 2019

Accepted Date: 2 December 2019

Please cite this article as: Esposito, F., Sechi, M., Pala, N., Sanna, A., Koneru, P.C., Kvaratskhelia, M., Naesens, L., Corona, A., Grandi, N., di Santo, R., D'Amore, V.M., Di Leva, F.S., Novellino, E., Cosconati, S., Tramontano, E., Discovery of dihydroxyindole-2-carboxylic acid derivatives as dual allosteric HIV-1 Integrase and Reverse Transcriptase associated Ribonuclease H inhibitors, Antiviral Research (2020), doi: https://doi.org/10.1016/j.antiviral.2019.104671. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Discovery of dihydroxyindole-2-carboxylic acid derivatives as dual allosteric HIV-1 Integrase and Reverse Transcriptase associated Ribonuclease H inhibitors. Francesca Esposito1*, Mario Sechi2, Nicolino Pala2, Adele Sanna2, Pratibha Chowdary Koneru3, Mamuka Kvaratskhelia3, Lieve Naesens4, Angela Corona1, Nicole Grandi1, Roberto di Santo5, Vincenzo Maria D’Amore6, Francesco Saverio Di Leva6, Ettore Novellino6, Sandro Cosconati7, and Enzo Tramontano1

1

Department of Life and Environmental Sciences, University of Cagliari, Cittadella Universitaria

SS554, 09042 Monserrato (CA), Italy; 2

Department of Chemistry and Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari, Italy;

3

Division of Infectious Diseases, University of Colorado School of Medicine, Aurora, CO 80045,

USA; 4

Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium;

5

Department of Drug Chemistry and Technologies, Istituto Pasteur-Fondazione Cenci Bolognetti,

“Sapienza” Università di Roma, Roma, Italy 6

Department of Pharmacy, University of Naples Federico II, Via D. Montesano 49, 80131 Naples,

Italy 7

DiSTABiF, University of Campania Luigi Vanvitelli, Via Vivaldi, 43, 81100 Caserta, Italy.

Corresponding Author* Dr. Francesca Esposito Department of Life and Environmental Sciences University of Cagliari Cittadella Universitaria di Monserrato SS554 09042 Monserrato (Cagliari) Italy tel +39-070-6754533 fax +39-070-6754536 e-mail [email protected]

Keywords: HIV dual inhibitors; IN; RNase H; IN-LEDGF binding inhibitors; sucrose binding site; dihydroxyindole-2-carboxylic acids.

Abstract The management of Human Immunodeficiency Virus type 1 (HIV-1) infection requires life-long treatment that is associated with chronic toxicity and possible selection of drug-resistant strains. A new opportunity for drug intervention is offered by antivirals that act as allosteric inhibitors targeting two viral functions (dual inhibitors). In this work, we investigated the effects of 5,6dihydroxyindole-2-carboxylic acid (DHICA) derivatives on both HIV-1 Integrase (IN) and Reverse Transcriptase associated Ribonuclease H (RNase H) activities. Among the tested compounds, the dihydroxyindole-carboxamide 5 was able to inhibit in the low micromolar range (1-18 µM) multiple functions of IN, including functional IN-IN interactions, IN-LEDGF/p75 binding and IN catalytic activity. Docking and site-directed mutagenesis studies have suggested that compound 5 binds to a previously described HIV-1 IN allosteric pocket. These observations indicate that 5 is structurally and mechanistically distinct from the published allosteric HIV-1 IN inhibitors. Moreover, compound 5 also inhibited HIV-1 RNase H function, classifying this molecule as a dual HIV-1 IN and RNase H inhibitor able to impair the HIV-1 virus replication in cell culture. Overall, we identified a new scaffold as a suitable platform for the development of novel dual HIV-1 inhibitors.

1. Introduction Over 30 million people are currently infected worldwide with the Human Immunodeficiency Virus1 (HIV-1), the causative agent of the Acquired Immunodeficiency Syndrome (AIDS). Despite the successful development of antiretroviral therapies, there is a still need for the identification of new drugs with innovative mechanisms of action that can be effective against drug-resistant strains, whose incidence is increasing among treated and naïve patients (Schneider et al. 2016; Margot et al. 2017; Gupta et al. 2017). HIV-1 belongs to the Retroviridae family, characterized by a replication

cycle in which the genomic ssRNA is retrotranscribed into proviral dsDNA. This process is carried out by the virally encoded reverse transcriptase (RT) which is a multifunctional protein endowed with two main enzymatic functions: an RNA-dependent DNA polymerase (RDDP) activity, that catalyzes the formation of the RNA:DNA intermediate, and a Ribonuclease H (RNase H) activity, involved in the hydrolytic cleavage of the RNA strand of the RNA:DNA hybrid (Esposito et al. 2012). After reverse transcription, another virally encoded enzyme, integrase (IN), allows for the integration of the proviral dsDNA into host chromosomes by performing two different catalytic reactions, named 3’-processing and strand-transfer (Esposito & Tramontano 2013). In the cytoplasm, during 3’-processing, the two conserved nucleotides GT are removed from each 3’-end of the long terminal repeat (LTR) terminus of the viral DNA. Next, the strand-transfer reaction catalyzes the covalent ligation of the proviral dsDNA into the host genome. The gap-filling at the interfaces between viral and host DNA is then completed using the host DNA repair machinery (Cherepanov et al. 2011). HIV-1 IN is composed of three functional domains: i) the N-terminal domain (NTD), characterized by a highly conserved zinc-binding motif (Polard & Chandler 1995; Rice et al. 1996), which stabilizes the protein folding and allows proper IN multimerization (Wang et al. 2010; Zhao et al. 2012); ii) the catalytic core domain (CCD), containing the catalytic D64-D116-E152 motif, that coordinates the magnesium ions; iii) the C-terminal domain (CTD) that is involved in strong nonspecific DNA-binding of both viral and cellular DNA (Engelman et al. 1994; Puras Lutzke et al. 1994). All three domains are important for functional oligomerization of IN. HIV-1 IN functions indeed as a multimer and forms the stable synaptic complex (SSC) with the viral DNA ends (Passos et al. 2017). During virus replication, IN operates within the so-called pre-integration complex (PIC), a macromolecular assemblage consisting of viral DNA, viral proteins and a number of host cell proteins (Miller et al. 1997). After the nuclear import, PICs engage with chromatin through protein-protein and DNA-protein interactions (Farnet & Haseltine 1991; Miller et al. 1997). Among the cellular factors involved in the integration process into the host DNA, there is LEDGF/p75 (Cherepanov et al. 2003; Angela Ciuffi, Manuel Llano, Eric Poeschla, Christian Hoffmann, Jeremy Leipzig, Paul Shinn 2005; Busschots et al. 2005; Vanegas et al. 2005; Manuel Llano, Dyana T. Saenz, Anne Meehan, Phonphimon Wongthida, Mary Peretz, William H. Walker, Wulin Teo 2006) a nuclear protein that promotes tethering of the SSC to chromatin by establishing specific interactions between its IN binding domain (IBD) and to the V-shaped pocket at the CCD dimer interface (Lutzke & Plasterk 1998). IN is a highly suitable pharmacological target, and four drugs targeting the IN catalytic site have been approved so far (Al-Mawsawi & Neamati 2011; Tsiang et al. 2016). In addition,

allosteric HIV-1 IN inhibitors (ALLINIs), also referred to as LEDGINs or non-catalytic site IN inhibitors (NCINIs), have been reported (Christ et al. 2010; Tsiang et al. 2012; Kessl et al. 2012; Demeulemeester et al. 2014; Integrase et al. 2014; Fader et al. 2014). ALLINIs bind the IN-IN dimer interface at the principal LEDGF/p75 binding site and induce higher-order, aberrant IN multimerization and potently inhibit HIV-1 replication during virus particle maturation (Balakrishnan et al. 2013; Belete Ayele Desimmie et al. 2013; Jurado et al. 2013; Rouzic et al. 2013; Bel et al. 2014). In vitro, ALLINIs exhibit a multimodal mechanism of action by i) promoting higher-order, aberrant IN multimerization, ii) inhibiting IN binding to the viral RNA, iii) adversely affecting the IN-viral DNA assembly, and iv) interfering with the IN-LEDGF binding (Christ et al. 2010; Tsiang et al. 2012; Kessl et al. 2012; Belete A Desimmie et al. 2013; Kessl et al. 2016). More recently, the natural compound Kuwanon-L was found to inhibit IN through an alternative allosteric mechanism of action. Indeed, this compound was reported to bind to a cleft originally identified as the sucrose recognition pocket, which is located at the CCD dimer interface close to the ALLINIs binding site (Wielens et al. 2010; Tintori et al. 2015). In addition to IN, another very promising target is the RT-associated RNase H activity since it is essential for virus replication and represents the only virus-encoded enzymatic function for which no drug is presently in clinical trials (Corona et al. 2013; Corona, Esposito, et al. 2014). In this context, great attention is dedicated to metal-chelating compounds, able to inhibit structurally homologous metal-dependent enzymes through Mg2+ chelation in the active site, which could be active against both HIV-1 IN and RNase H functions (Carcelli et al. 2014; Corona, Di Leva, et al. 2014; Corona, Di Leva, et al. 2016; Carcelli et al. 2017). It has been reported that some compounds carrying a dihydroxy-carboxamide backbone inhibit influenza virus PA endonuclease (Pala et al. 2015) and also hepatitis C virus (Zoidis et al. 2016). In particular, a number of dihydroxyindole-2-carboxylic acid (DHICA) derivatives, which inhibit the catalytic site of influenza virus PA endonuclease in a metal ion-dependent manner (Pala et al. 2015), were also previously found to reduce the HIV-1 IN catalytic activities (M. Sechi et al. 2004). In addition, some indole-based compounds were reported to be HIV-1 IN allosteric inhibitors (Patel et al. 2016), while others were shown to inhibit both RT-associated RDDP and RNase H functions, acting as dual RT inhibitors in the low micromolar range (Distinto et al. 2012; Meleddu et al. 2015; Meleddu et al. 2016). In the present work, we selected and tested compounds from an in house DHICAs library for the ability of these compounds to inhibit HIV-1 IN and RNase H activities. The results showed that compound 5 possesses the best profile in inhibiting the functionally important IN-IN subunit exchange, the IN-LEDGF/p75 binding, the IN catalytic activities, the RT-associated RNase H

function, and the HIV-1 replication in cell culture. In silico molecular docking and mutagenesis studies suggested that 5 binds to IN in the same pocket of the natural compound Kuwanon-L (Esposito et al. 2015; Martini et al. 2017), while it binds to HIV-1 RT in an allosteric site close to the catalytic RNase H site, in the RNase H primer grip, close to the p66/p51 heterodimer interface (Bauman et al. 2013). Overall, the DHICA scaffold appears to be a relevant chemotype for the development of new allosteric inhibitors with dual activity against HIV-1 IN and RNase H.

1. Materials and Methods.

2.1 Chemicals. 2.1.1 Materials and methods. Compounds 1-17 (Figure 1) have been prepared following synthetic procedures previously reported by us (Mario Sechi et al. 2004; Pala et al. 2015). Elemental

analyses for tested compounds were performed on a Perkin-Elmer 2400 spectrometer, and were within ±0.4% of the theoretical values, thus confirming ≥ 95% purity. 2.1.2 Chemistry. All solvents and other reagents were obtained from Aldrich, Merck or Carlo Erba. All reactions involving air- or moisture-sensitive compounds were performed under nitrogen atmosphere using oven-dried glassware and syringes to transfer solutions. Melting points (Mp) were determined using an electrothermal melting point or a Köfler apparatus and are uncorrected. Nuclear magnetic resonance (1H-NMR) spectra were determined in CDCl3 and DMSO-d6 on 400 MHz Bruker Advance III spectrometer. Chemical shifts (δ scale) are reported in parts per million (ppm) downfield from tetramethylsilane (TMS) used as an internal standard. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O. Mass spectra were obtained on a Hewlett-Packard 5989 Mass Engine Spectrometer, or a MALDI micro MX (Waters, Micromass) equipped with a reflectron analyzer. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel F-254 plates. Flash chromatography purifications were performed on Merck Silica gel 60 (230−400 mesh ASTM) as a stationary phase. Elemental analyses for tested compounds were performed on a PerkinElmer Elemental Analyzer 2400-CHN at Laboratorio di Microanalisi, Dipartimento di Chimica, Università di Sassari (Italy), and were within ±0.4% of the theoretical values.

2.2 Expression and purification of recombinant INs and LEDGFs. Recombinant 6xHis tagged wt, A128T, and H171T IN proteins were expressed and purified as described previously (Mckee et

al. 2008; Kessl et al. 2012; Feng et al. 2013; Slaughter et al. 2014; Esposito et al. 2016). Briefly, the pKBIN6Hthr plasmids containing wt IN sequence or the A128T and H171T single amino acid substitutions introduced by PCR were used to express the proteins in Escherichia coli strain BL21 (DE3). Initial purification was done using a Ni-Sepharose column with an imidazole gradient from 20 mM to 500 mM concentration in a 50 mM HEPES (pH 7.5) buffer containing 1 M NaCl, 7.5 mM CHAPS, 2 mM β-mercaptoethanol. This was followed by a heparin column purification with a NaCl gradient from 0 to 1M concentrations. FLAG-IN was purified by loading precipitate of cell lysate onto a Phenyl-sepharose and ammonium sulfate gradient (0 mM to 800 mM) in a 50 mM HEPES (pH 7.5) buffer containing 200 mM NaCl, 7.5 mM CHAPS, 2 mM β-mercaptoethanol. Peak fractions were pooled and loaded onto a heparin column and were eluted with an increasing NaCl gradient (200 mM to 1 M) in a 50 mM HEPES (pH 7.5) buffer containing 7.5 mM CHAPS and 2 mM β-mercaptoethanol. Fractions containing IN were pooled and stored in 15% glycerol at -80 °C. His-LEDGF and FLAG-LEDGF were purified by loading precipitate of cell lysate onto a heparin column and was eluted with an increasing NaCl gradient (200 mM to 1 M) in a 50 mM HEPES (pH 7.5) buffer containing 7.5 mM CHAPS and 2 mM β-mercaptoethanol. Peak fractions were pooled and loaded onto a Superdex 200 GL column and eluted in a buffer containing 50 mM HEPES (pH 7.5), 200 mM NaCl and 2 mM β-mercaptoethanol. Fractions containing LEDGF were pooled and stored in 15% glycerol at -80 °C. 2.3 Site-directed mutagenesis on HIV-1 RTs. Amino acid substitutions were introduced into the p66 HIV-1 RT subunit of an HIV-1 RT using a QuikChange mutagenesis kit, following the manufacturer's instructions (Agilent Technologies Inc., Santa Clara, CA). 2.4 Expression and purification of recombinant HIV-1 RTs. His-tagged p66/p51 HIV-1 RTs, wt or containing L503F or W535A substitutions were expressed in E. coli strain M15 (Rocca et al. 2019). Protein purification was carried out with a BioLogic LP system (Bio-Rad) with a combination of immobilized metal ion affinity chromatography and ion-exchange chromatography. Cell pellets were resuspended in lysis buffer containing 50 mM Sodium phosphate (pH 7.8) and 0.5 mg/ml lysozyme, the mixture was incubated on ice for 20 min, 0.3 M NaCl (final concentration) was added, and then, was sonicated and centrifuged for 1 h at 30,000 x g. The supernatant was loaded onto a Ni2+-Sepharose column that was pre-equilibrated with loading buffer, composed to 50 mM sodium phosphate (pH 7.8), 0.3 M NaCl, 15% glycerol, 15 mM imidazole and was washed thoroughly with wash buffer (50 mM sodium phosphate (pH 6.0), 0.3 M NaCl, 15% glycerol, 80 mM imidazole). RT was gradient eluted with elution buffer (wash buffer with 0.5 M imidazole). Enzyme-containing fractions were pooled, diluted 1:1 with dilution buffer (50 mM sodium

phosphate [pH 7.0], 15% glycerol), and then loaded onto a HiTrap Heparin HP column. The column was then washed with loading buffer 2, and RT was subjected to gradient elution with elution buffer 2 (50 mM sodium phosphate [pH 7.0], 15% glycerol, 150 mM NaCl). Purified protein was dialyzed and stored in buffer containing 50 mM Tris HCl (pH 7.0), 25 mM NaCl, 1 mM EDTA, and 50% glycerol. Enzyme-containing fractions were pooled, and aliquots were stored at -80 °C.

2.5 HTRF LEDGF-dependent and -independent assays. The IN LEDGF/p75-dependent assay allows measuring the inhibition of 3’-processing and strand-transfer IN reactions in the presence of recombinant LEDGF/p75 protein (Kessl et al. 2012; Feng et al. 2013; Esposito et al. 2015). Briefly, 50 nM IN was pre-incubated with increasing concentration of compounds for 1 hour at room temperature in reaction buffer containing 20 mM HEPES pH 7.5, 1 mM DTT, 1% Glycerol, 20 mM MgCl2, 0.05% Brij-35 and 0.1 mg/ml BSA. To this mixture, 9 nM DNA donor substrate (5’ACAGGCCTAGCACGCGTCG-Biotin-3’

annealed

with

5’-CGACGCGTGGTAGGCCTGT-

Biotin3’) and 50 nM DNA acceptor substrate (5’-Cy5-ATGTGGAAAATCTCTAGCAGT-3’ annealed with 5’-Cy5- TGAGCTCGAGATTTTCCACAT-3’) and 50 nM LEDGF/p75 protein (or without LEDGF/p75 protein) were added and incubated at 37 °C for 90 minutes. After the incubation, 4 nM of Europium-Streptavidin were added at the reaction mixture and the HTRF signal was recorded using a Perkin Elmer Victor 3 plate reader using a 314 nm for excitation wavelength and 668 and 620 nm for the wavelength of the acceptor and the donor substrates emission, respectively.

2.6 The HTRF-based IN-LEDGF binding assay. His-IN was pre-incubated with different concentrations of the test compounds in a buffer containing 150 mM NaCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mg/ml BSA, 25 mM Tris (pH 7.4) for 30 minutes at room temperature (Kessl et al. 2012; Esposito et al. 2015). Then, FLAG-LEDGF was added to the reaction and a mixture of antiHis6-XL665 and anti-FLAG-EuCryptate antibodies were then added to the reaction. After 4 hours at 4 °C, the HTRF signal was recorded using a Perkin Elmer Victor 3 plate reader using 314 nm for excitation wavelength and 668 and 620 nm for the wavelength of the acceptor and donor emission, respectively. The HTRF signal is defined as the emission ratio 665 nm/620 nm multiplied by 10,000.

2.7 The HTRF-based assay to monitor higher-order, aberrant IN multimerization or IN-IN Subunit Exchange. His and FLAG-tagged INs were mixed in 25 mM Tris (pH 7.4) buffer containing 150 mM NaCl, 2 mM MgCl2, 0.1% Nonidet P-40, 1 mg/ml BSA (Kessl et al. 2012;

Esposito et al. 2015). Test compounds were then added to the mixture and incubated for 2.5 hours at room temperature. A mixture of anti-His6-XL665 and anti-FLAG-EuCryptate antibodies were then added to the reaction and incubated at room temperature for 3 hours. The HTRF signal was recorded as above. The signal increase indicates higher-order, aberrant IN multimerization as seen with ALLINIs (Kessl et al. 2012). In contrast, the signal decrease indicates that a test compound interferes with IN-IN subunit exchange or functional IN-IN interactions.

2.8 RNase H polymerase-independent cleavage assay. The HIV-1 RT-associated RNase H activity was measured in 100 µL reaction volume containing 50 mM Tris HCl pH 7.8, 6 mM MgCl2, 1 mM DTT, 80 mM KCl, hybrid RNA/DNA (5’-GTTTTCTTTTCCCCCCTGAC-3’Fluorescein, 5’-CAAAAGAAAAGGGGGGACUG-3’-Dabcyl) and 3.8 nM RT. The reaction mixture was incubated for 1 hr at 37 °C, the reaction was stopped by addition of EDTA and products were measured with a Victor 3 (Perkin) at 490/528 nm (Distinto et al. 2012). The Yonetani-Theorell analysis was performed as previously reported (Massari et al. 2019). HIV-1 RT mutants were tested as described (Poongavanam et al. 2018), data were analyzed and figures were made with GraphPad Prism 6 version 6.01.

2.9 HIV-1 RT-associated RDDP assay. The HIV-1 RT-associated RDDP activity was measured using the Enz-Check Reverse Transcriptase Assay Kit, as previously described (Tintori et al. 2016).

2.10 Evaluation of MgCl2 chelation. Compounds were solubilized in 1 mL of 15% ethanol and 15 mM Tris HCl pH 7.8. The UV–VIS spectrum was recorded, from 250 nm to 600 nm, before and after the addition of 6 mM MgCl2 (Esposito et al. 2013).

2.11 Determination of anti-HIV-1 activity in MT4 cells. The method to determine the compounds anti-HIV activity and cytotoxicity in human lymphocyte MT-4 cells was reported elsewhere (Pannecouque et al. 2008). Briefly, serial compound dilutions were added to 96-well plates containing MT-4 cells. To virus-infected wells, HIV-1 (strain IIIB) was added at 100–300 CCID50 (50% cell culture infectious dose) per well. Mock-infected wells received the compounds without the virus. After five days of incubation, the effect of the compound on the viability of the mock- and HIV-infected cells was determined by the spectrophotometric MTT method. The 50% cytotoxic concentration (CC50) was defined as the concentration that reduced the viability of mock-infected MT-4 cells by 50%. The 50% effective concentration (EC50) was defined as the concentration achieving 50% protection from virus-induced cytopathic effect.

2.12 Molecular Modeling. X-ray structures selection. For IN, we selected the crystal structure of the enzyme CCD dimer with an inhibitor bound at an allosteric pocket that is close to the LEDGF binding site (PDB code: 3NF7)(Rhodes et al. 2011). Indeed, in this structure, the side chains of the residues shaping the sucrose binding pocket are arranged so as to host ligands of various sizes and nature, contrarily to what happens for the X-ray structure of the CCD dimer in complex with the sugar where the access to the cleft is partly hampered(Wielens et al. 2010). For RNase H, we chose the only crystal structure of the HIV-1 RT in complex with a ligand bound to the 428-507 site(Bauman et al. 2013) available in the Protein Data Bank (PDB code: 4IG0). Protein and Ligand Preparation. Protein structures were prepared using the “Protein Preparation Wizard” panel of the Schrödinger molecular modeling package. First, bond orders were assigned and all the hydrogen atoms were added. A prediction of the ionization and tautomeric states of the amino acids side chains was then carried out using Epik (Shelley et al. 2007; Greenwood et al. 2010). Optimization of the hydrogen-bonding network followed by a restrained minimization on hydrogen atoms was then performed. Finally, all the water molecules were deleted. The ligand tridimensional structure was generated with Maestro(Schrödinger 2019) and prepared for docking using Ligprep (Schrödinger, LLC 2019). Protonation states were predicted using Epik (Shelley et al. 2007; Greenwood et al. 2010). Molecular Docking. The AutoDock4.2 (AD4) software package(Morris et al. 2009) was used to perform molecular docking calculations. Using the software Autogrid 4.2, grid points of 60×60×50 with a 0.375Å spacing were calculated around the sucrose binding cavity of the IN CCD dimer, while for RT, a grid box of 50×50×50 points centered at the 428-507 site was set. For each system, 100 separate docking runs were performed. Each docking run consisted of 10 million energy evaluations using the Lamarckian genetic algorithm local search (GALS) method. Otherwise, default docking parameters were applied. Docking conformations were clustered on the basis of their r.m.s.d. (tolerance=2.0 Å) and were ranked based on the AutoDock scoring function (Huey et al. 2007). All the figures were rendered using PyMOL (http://www.pymol.org).

2. Results.

2.1 Effect of DHICA derivatives on HIV-1 IN activity in the presence of LEDGF/p75 cofactor An in house library containing several DHICA analogs (1-17, Figure 1) was tested for the ability to inhibit HIV-1 IN function in the presence of the LEDGF/p75 cofactor, using the strand transfer inhibitor raltegravir and the allosteric inhibitor Kuwanon-L (Esposito et al. 2015) as positive controls (Table 1). Among the tested compounds, 1 inhibited HIV-1 IN activity with an IC50 value of 7 µM, while its dimethoxy derivative 2 turned out as a very weak IN inhibitor. Also, the diketoester derivative 3 showed a weak ability to inhibit HIV-1 IN activity in the presence of LEDGF/p75 (IC50 value = 49.5 µM), while the corresponding acid 4 showed a higher IN inhibitory potency (IC50 value = 9 µM). The introduction of a dihydroxyphenethyl moiety in 1 afforded compound 5 that was able to potently inhibit LEDGF/p75 mediated HIV-IN activity (IC50 = 1.4 µM). However, the introduction of dioxole in 5, combined with other modifications such as the replacement of the ethyl catechol function with benzyl or phenethyl moieties, variously substituted with dioxole, dimethoxy, and trimethoxy groups (i.e. compounds 6, 7, 8, 9, 10, and 11), dramatically abolished the HIV-1 IN inhibitory activity. The dimerization of 1 through a carboxamido-propyl group linker led to compound 12, which also potently inhibited LEDGF/p75-dependent IN activity, with an IC50 value of 2.1 µM. In compound 12, the replacement of the hydroxyl groups with a dioxole and/or the elongation of the linker led to compounds 13-15 that were devoid of HIV-1 IN inhibition ability. On the other hand, 16, that presents a bis-propyl-piperazinyl linker produced a significant inhibition of the HIV-1 IN activity, with an IC50 value of 7 µM. Conversely, the dioxole derivative of 16, compound 17, resulted to be inactive.

2.2.2 Characterization of the mode of IN inhibition by DHICA derivatives. To gain insights into the mechanism of action of the selected DHICA derivatives, we preliminarily investigated whether these compounds were able to chelate the divalent metal ion cofactor, similar to what was already shown for the diketo acid derivatives (Tramontano et al. 2005; Costi et al. 2013; Carcelli et al. 2014; Corona, Schneider, et al. 2014; Cuzzucoli Crucitti et al. 2015; Corona, Di Leva, et al. 2016; Poongavanam et al. 2018). Hence, we measured their absorbance profile in the absence/presence of MgCl2. Interestingly, all the compounds were unable to chelate Mg2+ (data not shown), suggesting an alternative, potentially allosteric mode of action rather than a direct interaction with the IN orthosteric site. Subsequently, the most active compounds (1, 5, 12, and 16) in IN inhibition assays were tested for their ability to inhibit the IN-LEDGF/p75 binding, using Kuwanon-L as a positive control. The

results showed that 1 and 5 were able to inhibit the IN-LEDGF/p75 binding with an IC50 value of 15 and 18 µM, respectively (Table 2), whereas 12 turned out as the most effective the INLEDGF/p75 binding inhibitor, with an IC50 value of 0.18 µM. Conversely, compound 16 weakly inhibited the IN-LEDGF/p75 binding. In addition, we evaluated how these compounds affected IN activity in the absence of LEDGF/p75 (Table 2). Remarkably, compounds 5 and 12 were found to inhibit the HIV-1 IN strand-transfer catalytic activity both in the presence and absence of LEDGF/p75 protein with comparable IC50 values (Tables 1 and 2), while the IN inhibitory potency of compounds 1 and 16 decreased by 2- to 3-folds in the absence of LEDGF/p75. To gain further information on their mode of action, we tested the ability of these compound to affect the functional multimerization of IN. To this aim, we employed an HTRF-based assay which allows monitoring the interactions between full-length INs fused to either His or Flag tag (Kessl et al. 2012). In fact, compounds that reduce functional IN-IN interactions can be readily discriminated from those inducing higher-order, aberrant IN multimerization by yielding decreased versus increased HTRF signals, respectively. Data showed that all the tested compounds (1, 5, 12 and 16) inhibited IN-IN subunit exchange, whereas they failed to induce higher-order, aberrant IN multimerization (Table 2). In particular, compounds 12 and 16 modulated the IN-IN interaction in the submicromolar range (IC50 = 0.8 µM) and with an IC50 value of 1.75 µM, respectively, while compounds 1 and 5 showed IC50 values for IN-IN subunit exchange of ~15-20 µM.

2.2.3 Effect of DHICA derivatives in cell-based anti-HIV-1 assays. In order to determine their antiviral efficacy, compounds 1, 5, 12 and 16 were tested for HIV-1 inhibition and cytotoxicity in MT4 cells using nevirapine, zidovudine, and lamivudine as positive controls (Table 3). Results showed that compound 1 displays favorable antiviral activity with an EC50 value of 12 µM and a selectivity index (ratio of cytotoxic to antiviral concentration) of 6. Compound 5 was also active on replication with an EC50 value of 46 µM and a selectivity index of 3. On the other hand, compounds 12 and 16 were inactive.

2.2.4 Dissecting the mode of IN inhibition of compound 5 To further investigate the mode of action of our DHICAs we focused on the most potent compound in biochemical assays, compound 5, firstly testing it against HIV-1 INs A128T and H171T mutants. Data showed that compound 5 is 2- and 2.6-fold less active against the HIV-1 A128T and H171T IN mutants, respectively, compared to the wt-enzyme (Table 4). On the other hand, the HIV-1 A128T and H171T substitutions conferred 7.5- and 4-fold resistance to the compound ALLINI-2,

used as a control (Fadel et al. 2014). These data suggest that mutations A128T and H171T have different effects on the binding to IN of compound 5 compared to ALLINI-2. Secondly, since compound 5 was shown not to chelate Mg2+ and to act differently from ALLINI derivatives, we then verified whether it could bind to the same allosteric site proposed for the natural compound Kuwanon-L (Esposito et al. 2015; Martini et al. 2017). Hence, starting from the evidence that Kuwanon-L is antagonized by sucrose, we evaluated the inhibitory activity of compound 5 in the presence of 300 mM sucrose. Results showed that the addition of sucrose reduced the inhibitory effect by a 3 factor (Figure 2). To further verify the hypothesis that compound 5 could bind to the same pocket proposed for Kuwanon-L, we performed a combination inhibition study between the latter compound and 5, expressing the results with an isobologram plot (Figure 3). Data showed that 5 and Kuwanon-L are antagonistic for LEDGF/p75-dependent IN inhibition, suggesting that the two compounds reciprocally hamper their binding to IN. Results obtained with the isobologram plot were also supported by the fractional inhibitory concentration (FIC) value that was around 2. In fact, a FIC value higher than 1 indicates that the two compounds have antagonist effects (Martin et al. 1995).

2.2.5 Docking studies of compound 5 on IN. To gain insights into the binding mode of 5 to IN, docking calculations of this compound were performed at the sucrose binding pocket of the enzyme, which is placed at the interface between two CCDs in the proximity of the LEDGF binding site. Interestingly, in the lowest energy docking pose (Figure 4A), compound 5 establishes multiple polar contacts with amino acids of both the IN CCD units. In particular, the ligand can form, through its catechol ring, H-bonds with side chains of E85 of one CCD monomer and with R107 of the other one. Here, additional hydrogen bonds are established by the ligand dihydroxyindole-carboxamide moiety with the V88 backbone NH and the E96 carboxylate group. Finally, 5 can form cation-π interactions with the side chains of K103 and R107 of both IN CCDs, which further stabilize the ligand binding mode. Interestingly, the docking pose of 5 is superimposable to that of sucrose, however, the DHICA ligand penetrates deeper into the targeted cleft compared to the sugar (Figure 4B).

2.2.6 Effect of DHICA derivatives on both HIV-1 RT-associated RDDP and RNase H activities. A number of indole-based derivatives were previously shown to inhibit both RT-associated functions (Distinto et al. 2012; Tocco et al. 2013; Meleddu et al. 2015; Corona, Meleddu, et al. 2016); thus, we verified whether this effect could be also observed in the case of our DHICA

derivatives. As reference compounds, we included both the diketoacid inhibitor RDS1759 (Corona, Di Leva, et al. 2014) and the non-nucleoside RT inhibitor efavirenz (Table 5). The experiments showed that 1 can inhibit HIV-1 RT-associated RNase H activity with an IC50 value of 29 µM, while it is 2.9-fold less potent against the RT-associated RDDP activity. Differently, compound 5 showed an IC50 value of 17 µM against the RNase H activity, while showing no effect on the RDDP activity. Finally, compound 12 inhibited both HIV-1 RNase H and RDDP activities with the same potency (IC50 values of 29 and 37 µM, respectively), while 16 was not active on RT.

2.2.7 Characterization of compound 5 binding to RT The RT activities inhibition assays showed that 5 possesses the highest selectivity towards the RNase H function. Therefore, we decided to investigate in more detail the RNase H inhibition mechanism of this compound. First, we verified whether, even if devoid of metal chelation properties, 5 could bind to the RNase H active site. To this aim, we assayed the RNase H inhibition rate in the contemporaneous presence of increasing concentrations of compound 5 and of the metal chelator diketo acid derivative RDS1759 which we previously reported to bind in the RNase H catalytic pocket (Corona, Di Leva, et al. 2014). Results drawn according to the Yonetani-Theorell plot (Yonetani et al. 1982) showed that compound 5 and RDS1759 are kinetically mutually exclusive (Figure 5), demonstrating that the binding of compound 5 to RNase H hampers that of RDS1759, and vice versa. This suggested that 5 could bind to an allosteric cleft which is close to the RNase H orthosteric site. Interestingly, previous studies demonstrated the existence of a large polar amphipathic cleft located at the interface between the p51 and p66 subunits in the proximity of the RNase H catalytic domain, referred to as the 428-507 site (Bauman et al. 2013). Therefore, we hypothesized that 5 could bind to this site in the HIV-1 RNase H domain and accordingly performed docking calculations in the X-ray structure of the p51/p66 RT dimer in which a fragment ligand is shown to bind to this cleft (PDB code 4IG0) (Bauman et al. 2013). In the lowest energy docking pose (Figure 6), compound 5 can establish multiple favorable contacts with residues of both RT subunits. In particular, the ligand contacts p66 subunit through H-bonds formed by i) the catechol ring with the E404 side chain, ii) the carboxamide moiety with the Q507 side chain and iii) the dihydroxyindole ring with the L533 backbone. Notably, the latter group can engage an additional H-bond with the amide function of N255 of the p51 subunit. The ligand binding mode is further stabilized by van der Waals interactions with the L425 (p51) and L503 (p66) side chains and T-shape interactions with W426 (p51) and W535 (p66). Indeed, N255, L425, W426, and W535 are conserved residues among the HIV-1 infected patients (Alcaro et al. 2010). Further support to this binding hypothesis was given by a comparison between the predicted docking pose of 5 and the co-

crystallized RT fragment ligand (Bauman et al. 2013) in which the two molecules are virtually superimposable; moreover, a common recognition pattern could be identified in which both ligands are able to engage H-bonds with the side chains of residues K255 (p51) and Q507 (p66).

2.2.8 Effect of compound 5 on HIV-1 RT mutants In order to experimentally verify the binding mode suggested by computational studies, we introduced single substitutions into the 428-507 binding pocket. We then tested the impact of the substitutions of L503(p66) with phenylalanine and W535(p66) with alanine on the enzyme susceptibility to the inhibition by compound 5, using RDS1759 as control (Corona, Di Leva, et al. 2014) (Figure 7). Results showed that L503F substitution reduces the RNase H inhibitory potency of compound 5 by 3.7 folds (Table 6). More remarkably, the W535A mutation completely compromises the activity of the compound, with an impact greater than that observed for RDS1759 which in fact retained some residual activity (Figure 7).

Discussion

DHICA derivatives were previously reported to inhibit both the HIV-1 IN and the influenza virus PA endonuclease catalytic activity (Sechi M et al. 2004; Pala et al. 2015). The compounds assayed in the present work were selected by considering specific chemical modifications of the DHICA scaffold (Figure 8) such as i) the presence of free and protected hydroxy groups on the indole ring; ii) the introduction of a beta-diketo acid moiety; iii) the methylation of the indole nitrogen; iv) the amidation with substituted benzyl- or phenylethylamine groups. Also, variously functionalized DHICA dimers (12-17, Figure 8) containing linkers of different lengths were resynthesized. In particular, compound 1 showed a good potency of inhibition of HIV-1 IN with respect to its dimethoxy derivative (compound 2), which did not show inhibitory ability. In agreement with what reported in the literature (Long et al. 2004), derivative 3 showed a weak ability to inhibit HIV-1 IN activity in the presence of LEDGF/p75, while the corresponding acid 4, showed a higher IN inhibitory potency. The introduction of a hydroxyphenethyl moiety in compound 1 led to compound 5 that possess a high IN inhibitory activity, while the introduction in compound 5 of a dioxole with different substitutions completely abolished the HIV-1 IN inhibitory activity. Concerning derivatives 12-17, dimerization of 1 through a carboxamido-propyl group linker led to compound 12, which also potently inhibited the LEDGF/p75-dependent IN activity, while the conversion of the hydroxyl groups of 12 into a dioxole ring (compound 13); the homologation of the alkyl linker

(compound 14) produced instead a complete loss of HIV-1 IN inhibition. The same behavior was observed for compound 15 where the two indole rings are linked through a hexyl fragment. Conversely, different linker such as a bis-propyl-piperazine- (compound 16) produced a significative inhibition of HIV-1 IN activity, where the corresponding dioxole derivative 17 resulted to be inactive. In order to characterize the mechanism of action of the most active compounds, they were tested for their ability to inhibit IN-LEDGF/p75 binding. Similarly to the positive control Kuwanon-L, all tested compounds were able to inhibit the IN-LEDGF/p75 binding, with 12 that turned out as the most effective compound in this assay. In addition, similarly to Kuwanon-L, compounds 5 and 12 were found to inhibit the HIV-1 IN strand-transfer catalytic activity both in the presence and absence of LEDGF/p75 protein, differently from compounds 1 and 16 which show a decrease in their potency of inhibition. Results showed that compounds 1, 5, 12 and 16 modulated the IN-IN interaction, and, in particular, compound 12 and 16 in a low micromolar range. Of note, DHICA tested derivatives showed a different profile of interference on the IN-IN interaction with respect to the one reported for Kuwanon-L. In fact, Kuwanon-L was able to induce higher-order, aberrant IN multimerization, while DHICA derivatives reduced the IN-IN subunit exchange, suggesting differences in the mode of action of DHICAs with respect to Kuwanon-L. In addition, compound 5 was also tested against HIV-1 INs A128T and H171T mutants. In fact, A128 and H171 are located at the HIV-1 IN dimer interface and were reported to confer resistance to certain ALLINIs (Christ et al. 2010; Tsiang et al. 2012; Feng et al. 2013; Slaughter et al. 2014). The data showed that these mutations have a different impact on the binding of compound 5 and compound ALLINI-2 further suggesting that DHICA derivatives bind differently from ALLINIs. To better understand the binding mode of these DHICA derivatives to HIV-1 IN, and since the compounds are not able to chelate Mg2+, we wanted to verify if compound 5, chosen since it was the most potent in biochemical assays among the compounds able to inhibit viral replication, could compete with the allosteric compounds Kuwanon-L. Biochemical studies performed in the presence of compound 5 and sucrose revealed that sugar reduced the IN inhibitory potency of 5 by 3 folds, suggesting that sucrose competes with this compound for binding to IN. In addition, also the presence of Kuwanon-L was shown to reduce compound 5 efficacy on IN, further suggesting that 5 and Kuwanon-L bind to the same site. Docking studies supported this hypothesis, also showing additional hydrogen bonds that may be established by the dihydroxyindole-carboxamide moiety of 5 with the V88 backbone NH and the E96 carboxylate group compared to Kuwanon-L (Esposito et al. 2015). Of note, both 5 and Kuwanon-L exert modulatory effects on IN-IN interactions. However,

5 apparently destabilizes these contacts, reducing new IN-IN dimer formation, while Kuwanon-L seems to stabilize them, moving the IN-IN equilibrium towards the dimeric state. Hence, although the two compounds bind to the same site, they can differently modulate IN-IN interactions. In addition, as reported for some indole-based derivatives (Distinto et al. 2012; Meleddu et al. 2015; Corona, Meleddu, et al. 2016), compound 5 was shown to be active against the RT-associated RNase H activity. Although we found that 5 is not able to chelate Mg (data not shown), we could not exclude its binding into the RNase H domain. The Yonetani-Theorell plot (Yonetani et al. 1982) showed that compound 5 and the RNase H selective diketo acid derivative RDS1759 are kinetically mutually exclusive, demonstrating that the binding of compound 5 to RNase H alters the RDS1759 binding, and vice versa. This suggests that compound 5 might bind to a site close to the catalytic RNase H site in the RNase H primer grip or that 5 induces a conformational transition in the protein that is not conducive of an interaction with RDS1759. Docking studies on 5 suggest that this compound can establish multiple favorable contacts with residues of both RT subunits, especially with E404, L503, Q507, L533 and W535 in the p66 subunit and N255 in the p51 subunit. With the aim of evaluating the effect of the overall shape and steric hindrance of this cleft as well as the importance of stacking/lipophilic interactions on ligand binding, single point mutations were introduced into the 428-507 binding pocket, demonstrating that L503F substitution reduces the RNase H inhibitory potency, while the W535A mutation completely compromises the activity of the compound. These data are in agreement with the binding hypothesis of 5 to the 428-507 allosteric pocket, and of its interaction with highly conserved HIV-1 RT residues such as W535.

Conclusions In this study, we have identified compound 5, a novel DHICA derivative that is able to allosterically inhibit both the HIV-1 IN and RNase H functions. Compound 5 adversely affected functional IN-IN interactions as well as inhibited the IN-LEDGF/p75 binding, and, hence, it impaired the HIV-1 IN strand transfer catalytic reaction in the presence and absence of LEDGF/p75 protein. Docking calculations and biochemical data suggested that compound 5 binds to an IN site close to the ALLINI binding pocket, that was previously proposed for the natural compound Kuwanon-L. Future studies are necessary to better understand the structural and mechanistic details on the 5/IN interaction, in particular, the molecular basis for the different modulation of the IN function by compound 5 compared to other compounds such as Kuwanon-L which bind to the same site but differently affect the IN-IN interactions. In addition to IN inhibition, compound 5 inhibited the HIV-1 RT-associated RNase H function through the interaction with some conserved amino acid

residues (Alcaro et al. 2010), showing an interesting dual inhibitor profile which is different from other dual HIV-1 inhibitors. (Distinto et al. 2013; Corona, Di Leva, et al. 2016) Remarkably, 5 also demonstrated to suppress HIV-1 replication in a cell-based assay. Overall, the DHICA scaffold can be considered a novel and attractive structure for the development of new dual IN and RNase H agents having an allosteric mechanism of action that is innovative with respect to the anti-HIV-1 drugs developed so far.

Acknowledgments This work was supported in part by National Institutes of Health grants R01 AI110310 and U54 GM103368 (to M.K.).

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Figure captions.

Figure 1. Structures of compounds 1-17.

Figure 2. Effect of compound 5 and compound 5 plus sucrose on the HIV-1 IN activity in the HTRF LEDGF/p75 dependent assay. Curve fitting of dose-dependent inhibition of IN in presence of LEDGF/p75 of compound 5 alone ( ) or compound 5 combined with sucrose (o).

Figure 3. Isobologram plot of the combination of compound 5 and Kuwanon-L in HIV-1 IN LEDGF/p75 dependent assay.

Figure 4. (A) Docking pose of 5 at the sucrose binding pocket of the HIV-1 IN CCD dimer (PDB code: 3NF7). The ligand is shown as cyan sticks, while the two IN monomers are represented as yellow and purple cartoon and transparent surfaces. Amino acids important for ligand binding are highlighted as sticks. Nonpolar hydrogens are omitted for clarity. Hydrogen bonds are shown as dashed black lines. (B) Superposition between 5 (cyan sticks) and sucrose (pink sticks) at the sucrose binding pocket of the HIV-1 IN CCD dimer (PDB code: 3LV3).

Figure 5. Yonetani–Theorell analysis. Combination of compound 5 and RDS1759 on the HIV-1 RT RNase H activity. HIV-1 RT was incubated in the presence of compound RDS1759 alone ( with increasing concentrations of compound 5µM ( ), 10 µM(

) and 20 µM(

) or combined

).

Figure 6. (A) Docking pose of 5 at the 428-507 binding pocket of the HIV-1 RT (PDB code: 4IG0). The ligand is displayed as cyan sticks. The p51 and p66 subunits are depicted as light red and grey cartoons, respectively, with transparent surfaces. Receptor amino acids important for ligand binding are highlighted as sticks. (B) Superposition between 5 (cyan sticks) and the co-crystallized allosteric RT ligand (yellow sticks) at the 428-507 binding pocket of the HIV-1 p51/p66 dimer. Amino acids contacted by both ligands are highlighted as sticks. In both figures, hydrogen bonds are shown as dashed lines and nonpolar hydrogens are omitted for clarity.

Figure 7. Effect of compound 5 on the RNase H activity of HIV-1 RT mutants.

Figure 8. Structure of the title compound 1 and selection of its derivatives.

Table 1. DHICA derivatives inhibition of the HIV-1 IN LEDGF/p75-dependent activity.

Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Raltegravir Kuwanon-L a

a

IC50 (µM)

7.0 ± 0.5 > 100 (62%)b 49 ± 1.5 9.0 ± 2.0 1.4 ± 0.6 > 100 (98%) > 100 (83%) > 100 (100%) > 100 (100%) > 100 (100%) > 100 (96%) 2.1 ± 0.1 > 100 (100%) > 100 (100%) > 100 (100%) 7.0 ± 0.1 > 100 (96%) 0.058 ± 0.01 42 ± 3.0

Concentration required to inhibit the HIV-1 IN catalytic activities, in the presence of LEDGF, by 50%. b Percentage of control activity measured in the presence of 100 µM compound concentration. Data are the mean ± SD of 3 independent experiments.

Table 2. Effect of DHICA derivatives on HIV-1 IN/LEDGF binding, IN LEDGF-independent activity, IN-IN subunit exchange and IN multimerization.

Compd.

a

IC50 IN-LEDGF binding (µM)

b

IC50 IN LEDGFindependent (µM)

c

IC50 IN-IN subunit exchange (µM)

15 ± 1.1 20 ± 0.3 22 ± 2.5 1 18 ± 6.0 2.8 ± 0.1 16 ± 3.0 5 0.18 ± 0.02 1.5 ± 0.5 0.8 ± 0.2 12 55 ± 10 15 ± 3.0 1.7 ± 0.1 16 22 ± 0.5 34 ± 0.5 >100 (100%) Kuwanon-L a Compound concentration required to inhibit the HIV-1 IN LEDGF interaction by 50%. b Compound concentration required to inhibit the HIV-1 IN catalytic activities by 50% in the absence of LEDGF. c Compound concentration required to inhibit the HIV-1 IN-IN subunit exchange by 50%. d Compound concentration required to inhibit the multimerization increase by 50%. Data are the mean ± SD of 3 independent experiments.

d

MI50 IN multimerization (µM) >100 (100%) >100 (100%) >100 (100%) >100 (100%) 38 ± 0.02

Table 3. Inhibition of HIV-1 replication by DHICA derivatives.

Compound 1 5 12 15 16 Nevirapine Zidovudine Lamivudine a

a

EC50 (µM)(µM) 12 ± 2 46 ± 16 > 153 >171 > 176 0.31 ± 0.06 0.0071 ± 0.0029 2.2 ± 0.8

b

CC50 (µM) 75 ± 33 133 ± 16 154 ± 15 171 ± 16 176 ± 31 > 150 > 94 > 87

Compound concentration required to inhibit HIV-1 (strain IIIB) replication in MT4 cells by 50%. b Compound concentration required to inhibit MT4 cell viability by 50%. Data are the mean ± SD of 2-4 independent experiments

Table 4. Inhibition of HIV-1 mutated INs by compound 5.

a

Compound wt IN

a

IC50 (µM)

A128T IN

H171T IN b

5

1.4 ± 0.6

2.8 ± 0.5 (2)

3.6 ± 0.5 (2.6)

ALLINI-2

0.15 ± 0.01

1.16 ± 0.09 (7.5)

0.63 ± 0.02 (4)

Compound concentration required to inhibit the HIV-1 IN catalytic activities, in the presence of LEDGF, by 50%. b Numbers in parenthesis indicate the fold of increase of IC50 values obtained on mutant INs with respect to wt IN. Data are the mean ± SD of 3 independent experiments.

Table 5. Inhibition of HIV-1 RT-associated RDDP and RNase H activities by DHICA derivatives.

Compound

a

a

IC50 RNase H (µM)

b

IC50 RDDP (µM)

1

29 ± 2.8

82 ± 5.0

5 12 16 RDS 1759

17 ± 0.5 24 ± 1.6 > 100 (85%) 7.3 ± 0.1

> 100 (90%)c 37 ± 1.1 > 100 (90%) -

Efavirenz

-

0.012 ± 0.003

Compound concentration required to inhibit the HIV-1 RNase H activity by 50%. Compound concentration required to inhibit the HIV-1 RDDP activity by 50%. c Percentage of control measured in the presence of 100 µM concentration. Data are the mean ± SD of 3 independent experiments. b

Table 6. Effect of compound 5 on RT-associated RNase H activity of mutated HIV-1 RTs

Compdound

a

IC50 RNase H (µM)

wt RT L503F W535A b 17.5 ± 0.5 65 ± 10 (3.7) >100 (>5.7) 5 24.1 ± 1.9 >100 (>4.0) >100 (>4.0) RDS1759 a Concentration required to inhibit HIV-1 RT-associated RNase H activity by 50%. b Numbers in parenthesis indicate the fold of increase of IC50 values obtained on mutant RTs with respect to wt RT.Data are the mean ± SD of 3 independent experiments.

50

Kuwanon-L (uM)

40

30

20

10

0 0,0

0,2

0,4

0,6

0,8

Compound 5 (uM)

1,0

1,2

1,4

Highlights

5,6-dihydroxyindole-2-carboxylic acid derivatives are a new scaffold for the development of novel dual HIV-1 inhibitors. The dihydroxyindole-carboxamide compound 5 inhibits multiple functions of the HIV-1 IN in the low micromolar range. The dihydroxyindole-carboxamide compound 5 inhibits the HIV-1 virus replication in cell culture. Dihydroxyindole-carboxamide compound 5 has a mode of action different from the known allosteric HIV-1 IN inhibitors.