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Potent Inhibition of HIV-1 Replication by Novel Non-peptidyl Small Molecule Inhibitors of Protease Dimerization

2007; Elsevier BV; Volume: 282; Issue: 39 Linguagem: Inglês

10.1074/jbc.m703938200

1083-351X

Yasuhiro Koh, Shintaro Matsumi, Debananda Das, Masayuki Amano, David A. Davis, Jianfeng Li, Sofiya Leschenko, Abigail Baldridge, Tatsuo Shioda, Robert Yarchoan, Arun K. Ghosh, Hiroaki Mitsuya,

HIV/AIDS Research and Interventions

Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transfer-based HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 μm and blocked HIV-1 replication with IC50 values of 0.0002-0.48 μm. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1. Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transfer-based HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 μm and blocked HIV-1 replication with IC50 values of 0.0002-0.48 μm. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1. Highly active antiretroviral therapy has had a major impact on the AIDS epidemic in industrially advanced nations. However, eradication of human immunodeficiency virus, type 1 (HIV-1) 2The abbreviations used are: HIV-1human immunodeficiency virus, type 1FRETfluorescence resonance energy transferCFPcyan fluorescent proteinYFPyellow fluorescent proteinBCVbrecanavirDRVdarunavirCHXcycloheximidePIprotease inhibitorbis-THFbistetrahydrofuranylurethaneTPVtipranavirFlucfirefly luminescenceRlucRenilla luminescenceRTreverse transcriptasePRprotease. does not appear to be currently possible, in part due to the viral reservoirs remaining in blood and infected tissues. Moreover, a number of challenges have been encountered, which include various adverse effects, only partial and limited immunologic restorations achieved, and occurrence of various cancers as consequences of survival elongation with highly active antiretroviral therapy (1Simon V. Ho D.D. Nat. Rev. Microbiol. 2003; 1: 181-190Crossref PubMed Scopus (114) Google Scholar). Moreover, such limitations of highly active antiretroviral therapy are exacerbated by the development of drug-resistant HIV-1 variants (2Carr A. Nat. Rev. Drug Discov. 2003; 2: 624-634Crossref PubMed Scopus (170) Google Scholar). Thus, the identification of new classes of antiretroviral drugs that have one or more unique mechanisms of action and produce no or minimal adverse effects remains an important therapeutic objective. human immunodeficiency virus, type 1 fluorescence resonance energy transfer cyan fluorescent protein yellow fluorescent protein brecanavir darunavir cycloheximide protease inhibitor bistetrahydrofuranylurethane tipranavir firefly luminescence Renilla luminescence reverse transcriptase protease. Dimerization of HIV-1 protease subunits is an essential process for the acquisition of proteolytic activity of HIV-1 protease, which plays a critical role in the maturation and replication of the virus (3Wlodawer A. Miller M. Jaskolski M. Sathyanarayana B.K. Baldwin E. Weber I.T. Selk L.M. Clawson L. Schneider J. Kent S.B. Science. 1989; 245: 616-621Crossref PubMed Scopus (1051) Google Scholar, 4Kohl N.E. Emini E.A. Schleif W.A. Davis L.J. Heimbach J.C. Dixon R.A. Scolnick E.M. Sigal I.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4686-4690Crossref PubMed Scopus (1280) Google Scholar). Thus inhibition of protease dimerization by chemical reagents is likely to abolish proteolytic activity and inhibit HIV-1 replication. However, for possible development of HIV-1 protease dimerization inhibitors, better understanding of the nature and dynamics of protease dimerization is crucial. The monomer subunits are connected by polar and non-polar interactions to form the dimer. Hydrophobicity of Leu-89, Leu-90, and Ile-93 and several other residues have been considered important in the folding of a protease monomer as well as in dimer stabilization (5Lapatto R. Blundell T. Hemmings A. Overington J. Wilderspin A. Wood S. Merson J.R. Whittle P.J. Danley D.E. Geoghegan K.F. Hawrylik S.J. Lee S.E. Scheld K.G. Hobart P.M. Nature. 1989; 342: 299-302Crossref PubMed Scopus (411) Google Scholar, 6Spinelli S. Liu Q.Z. Alzari P.M. Hirel P.H. Poljak R.J. Biochimie (Paris). 1991; 73: 1391-1396Crossref PubMed Scopus (185) Google Scholar). For a systematic analysis of the conserved network of hydrogen bonds, termed “fireman's grip,” Strisovsky et al. (7Strisovsky K. Tessmer U. Langner J. Konvalinka J. Krausslich H.G. Protein Sci. 2000; 9: 1631-1641Crossref PubMed Scopus (39) Google Scholar) have mutated the active site Thr-26 to a Ser, Cys, or Ala and have shown that T26A substitution reduced protease dimer stability, thus virtually nullifying the proteolytic activity of protease. Indeed, in our present study, T26A substitution effectively disrupted protease dimerization (see below), corroborating the results by Strisovsky et al. The flexibility of monomeric and dimeric HIV-1 protease and the feasibility of a stable protease monomer have also been studied by computational simulation (8Levy Y. Caflisch A. Onuchic J.N. Wolynes P.G. J. Mol. Biol. 2004; 340: 67-79Crossref PubMed Scopus (89) Google Scholar, 9Levy Y. Caflisch A. J. Phys. Chem. B. 2003; 107: 3068-3079Crossref Scopus (41) Google Scholar). There are four anti-parallel β-sheets involving the N and C termini of both monomer subunits and they contribute close to 75% of the dimerization energy (10Todd M.J. Semo N. Freire E. J. Mol. Biol. 1998; 283: 475-488Crossref PubMed Scopus (161) Google Scholar), explaining at least in part why DRV failed to dissociate mature protease dimer (see below). The termini interface has been explored as a dimerization inhibition target by several groups (11Bowman M.J. Byrne S. Chmielewski J. Chem. Biol. 2005; 12: 439-444Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 12Frutos S. Rodriguez-Mias R.A. Madurga S. Collinet B. Reboud-Ravaux M. Ludevid D. Giralt E. Biopolymers. 2007; 88: 164-173Crossref PubMed Scopus (26) Google Scholar, 13Bannwarth L. Kessler A. Pethe S. Collinet B. Merabet N. Boggetto N. Sicsic S. Reboud-Ravaux M. Ongeri S. J. Med. Chem. 2006; 49: 4657-4664Crossref PubMed Scopus (31) Google Scholar). We have also recently reported that certain peptides containing the dimer interface sequences amino acids 1-5 and amino acids 95-99 blocked HIV-1 infectivity and replication (14Davis D.A. Brown C.A. Singer K.E. Wang V. Kaufman J. Stahl S.J. Wingfield P. Maeda K. Harada S. Yoshimura K. Kosalaraksa P. Mitsuya H. Yarchoan R. Antiviral Res. 2006; 72: 89-99Crossref PubMed Scopus (28) Google Scholar). However, to the best of our knowledge, no evidence of direct dimerization inhibition by such compounds has been yet documented. In the present study, we developed an intermolecular fluorescence resonance energy transfer (FRET)-based HIV-1-expression assay that employed cyan and yellow fluorescent protein-tagged HIV-1 protease monomers. Using this assay, we identified a group of non-peptidyl small molecule inhibitors of HIV-1 protease dimerization. These inhibitors, including the recently approved protease inhibitor (PI) darunavir (DRV) as well as two experimental protease inhibitors (PIs), blocked protease dimerization at concentrations of as low as 0.01 μm and blocked HIV-1 replication in vitro with IC50 values of 0.0002-0.48 μm. These agents also inhibited the proteolytic activity of mature HIV-1 protease. Another PI, tipranavir (TPV), active against HIV-1 variants resistant to multiple PIs, also blocked protease dimerization, although all other existing FDA-approved anti-HIV-1 drugs examined in the present study failed to block the dimerization. The present report represents the first demonstration that non-peptidic small molecule agents can disrupt protease dimerization. Generation of FRET-based HIV-1 Expression System—Cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged HIV-1 protease constructs were generated using BD Creator™ DNA cloning kits (BD Biosciences, San Jose, CA). First, XhoI/HindIII fragments from pCR-XL-TOPO vector containing the HIV-1 protease-encoding gene excised from pHIV-1NL4-3 was inserted into the pDNR-1r (donor vector) that had been digested with XhoI and HindIII. In the transfer of the protease gene from the donor vector into pLP-CFP/YFP-C1 (acceptor vector), the Cre-loxP site-specific recombination method was used according to manufacturer's instructions. Using Cre-recombinase with the lox P site, the protease gene from pDNR-1r was inserted into pLP-CFP-C1 or pLP-YFP-C1 through Cre-mediated recombination (15Hoess R.H. Abremski K. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1026-1029Crossref PubMed Scopus (156) Google Scholar), generating a plasmid of CFP-tagged wild type protease (PRWT) and that of YFP-tagged PRWT, with which HIV-1 protease was successfully expressed as a fusion protein with CFP- and YFP-tagged at the C terminus, respectively. Western blot assay using anti-green fluorescent protein-specific rabbit polyclonal antibodies revealed that protease was correctly tagged to CFP or YFP (data not shown). For the generation of full-length molecular infectious clones containing CFP- or YFP-tagged protease, the PCR-mediated recombination method was used (16Fang G. Weiser B. Visosky A. Moran T. Burger H. Nat. Med. 1999; 5: 239-242Crossref PubMed Scopus (48) Google Scholar). To this end, we amplified an upstream proviral DNA fragment containing ApaI site and HIV-1 protease (excised from pHIV-1NL4-3) with a primer pair: Apa-PRO-F (5′-TTG CAG GGC CCC TAG GAA AAA GG-3′) plus PR-5Ala-R (5′-GGC TGC TGC GGC AGC AAA ATT TAA AGT GCA GCC AAT CT-3′), a middle proviral DNA fragment containing CFP (excised from pCFP-C1) or YFP (excised from pYFP-C1) (Clontech, Mountain View, CA) with a primer pair: CFPYFP-5Ala-F (5′-GCT GCC GCA GCA GCC GTG AGC AAG GGC GAG GAG CTG-3′) plus CFPYFP-FP-R (5′-ACT AAT GGG AAA CTT GTA CAG CTC GTC CAT GCC G-3′), and a downstream proviral DNA fragment containing the 5′-DNA fragment of reverse transcriptase (RT) and SmaI site from pHIV-1NLSma (17Gatanaga H. Suzuki Y. Tsang H. Yoshimura K. Kavlick M.F. Nagashima K. Gorelick R.J. Mardy S. Tang C. Summers M.F. Mitsuya H. J. Biol. Chem. 2002; 277: 5952-5961Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), which had been created to have a SmaI site by changing two nucleotides (2590 and 2593) of pHIV-1NL4-3 with a primer pair: FRT-F (5′-TTT CCC ATT AGT CCT ATT GAG ACT GTA-3′) plus NL4-3-RT263-R (5′-CCA GAA ATC TTG AGT TCT CTT ATT-3′). A linker consisting of five alanines was inserted between protease and fluorescent protein. The phenylalanine-proline site that HIV-1 protease cleaves was also introduced between the fluorescent protein and RT. Thus obtained three DNA fragments were subsequently joined by using the PMR reaction performed under the standard condition for ExTaq polymerase (Takara Bio Inc., Otsu, Japan) with 10 pmol of Apa-PRO-F (5′-TTG CAG GGC CCC TAG GAA AAA GG-3′) and NL4-3-RT263-R (5′-CCA GAA ATC TTG AGT TCT CTT ATT-3′) and the three DNA fragments (100 ng each) in a 20-μl reaction solution. Thermal cycling was carried out as follows: 94 °C for 3 min, followed by 35 cycles of 94 °C for 50 s, 53 °C for 50 s, and 72 °C for 2 min, and finally by 72 °C for 15 min. The amplified PCR products were cloned into pCR-XL-TOPO vector according to the manufacturer's instructions (Gateway Cloning System, Invitrogen). PCR products were generated with pCR-XL-TOPO vector as templates, followed by digestion by both ApaI and SmaI, and the ApaI-SmaI fragment was introduced into pHIV-1NLSma (17Gatanaga H. Suzuki Y. Tsang H. Yoshimura K. Kavlick M.F. Nagashima K. Gorelick R.J. Mardy S. Tang C. Summers M.F. Mitsuya H. J. Biol. Chem. 2002; 277: 5952-5961Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), generating pHIV-PRWTCFP and pHIV-PRWTYFP respectively. Analysis of Inter- and Intra-molecule Interactions of Protease Subunits—Analysis of inter- and intra-molecule interactions of protease subunits was conducted by employing the crystal structure of DRV with HIV-1 protease (PDB ID: 2IEN). Hydrogens were added and minimized using the OPLS2005 force field with constraints on heavy atom positions. The calculation was performed using MacroModel 9.1 from Schrödinger, LLC. Hydrogen bonds were assigned when the following distance and angle cut-off was satisfied: 3.0 Å for H-A distance; D-H-A angle >90°; and H-A-B angle >60° where H is the hydrogen, A is the acceptor, D is the donor, and B is a neighbor atom bonded to the acceptor. The representative distance between the termini of two monomers was determined by analyzing the protease-DRV crystal structure (PDB ID: 2IEN). The distance between the α carbons at the N termini and C termini is around 0.5 nm, whereas the distance between the α carbons of the N termini ends of two monomers is around 1.8 nm. FRET Procedure—COS7 cells plated on EZ view cover-glass bottom culture plate (Iwaki, Tokyo) were transfected with the indicated plasmid constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions in the presence of various concentrations of each compound, cultured for 72 h, and analyzed under Fluoview FV500 confocal laser scanning microscope (Olympus Optical Corp., Tokyo) at room temperature. When the effect of each compound was analyzed by FRET, test compounds were added to the culture medium simultaneously with plasmid transfection. The results of FRET were determined by quenching of CFP (donor) fluorescence and an increase in YFP (acceptor) fluorescence (sensitized emission), because part of the energy of CFP is transferred to YFP instead of being emitted. This phenomenon can be measured by bleaching YFP, which should result in an increase in CFP fluorescence. This technique, also known as acceptor photobleaching, is a well established method of determining the occurrence of FRET (18Sekar R.B. Periasamy A. J. Cell Biol. 2003; 160: 629-633Crossref PubMed Scopus (657) Google Scholar, 19Bastiaens P.I. Majoul I.V. Verveer P.J. Soling H.D. Jovin T.M. EMBO J. 1996; 15: 4246-4253Crossref PubMed Scopus (242) Google Scholar, 20Bastiaens P.I. Jovin T.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8407-8412Crossref PubMed Scopus (152) Google Scholar, 21Szczesna-Skorupa E. Mallah B. Kemper B. J. Biol. Chem. 2003; 278: 31269-31276Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Dequenching of the donor CFP by selective photobleaching of the acceptor YFP was performed by first obtaining YFP and CFP images at the same focal plane, followed by illuminating for 3 min the same image at a wavelength of 488 nm with a laser power set at the maximum intensity to bleach YFP and re-capturing the same CFP and YFP images. The changes in the CFP and YFP fluorescence intensity in the images of selected regions were examined and quantified using Olympus FV500 Image software system (Olympus Optical Corp.). Background values were obtained from the regions where no cells were present and were subtracted from the values for the cells examined in all calculations. For each chimeric protein, the data were obtained from at least three independent experiments. Digitized image data obtained from the experiment were prepared for presentation using Photoshop 6.0 (Adobe Systems, Mountain View, CA). Ratios of intensities of CFP fluorescence after photobleaching to CFP fluorescence prior to photobleaching (CFPA/B ratios) were determined. It is well established that the CFPA/B ratios of >1.0 indicate that association of CFP- and YFP-tagged proteins occurred, and it was interpreted that the dimerization of protease subunits occurred. When the CFPA/B ratios were 1.0, it was determined that HIV-1 protease had been generated and dimerization had occurred. The production of HIV-1 was monitored every 24 h following transfection by determining levels of p24 Gag protein produced into culture medium as previously described (24Koh Y. Nakata H. Maeda K. Ogata H. Bilcer G. Devasamudram T. Kincaid J.F. Boross P. Wang Y.F. Tie Y. Volarath P. Gaddis L. Harrison R.W. Weber I.T. Ghosh A.K. Mitsuya H. Antimicrob. Agents Chemother. 2003; 47: 3123-3129Crossref PubMed Scopus (336) Google Scholar). Generation of FRET-based HIV-1 Expression Assay—The basic concepts of the intermolecular FRET-based HIV-1-expression assay (FRET-HIV-1 assay) are shown in Fig. 1. Within a plasmid (pHIV-1NL4-3), which encodes a full-length molecular infectious HIV-1 clone, the gene encoding a CFP was attached to the downstream end (C terminus) of the gene encoding an HIV-1 protease subunit through the flexible linker added (five alanines), generating pHIV-1NL4-3/CFP (Fig. 1A). Within the other plasmid (pHIV-1NL4-3), the gene encoding a YFP was attached to the downstream end of protease-encoding gene in the same manner, generating pHIV-1NL4-3/YFP. Both CFP and YFP were designed to have phenylalanine and proline in the connection with RT so that the protease is cleaved from RT when two subunits dimerize and the dimerized protease acquires enzymatic activity. Fig. 1B illustrates that HIV-1 virions generated in COS7 cells transfected with pHIV-1NL4-3/CFP contained CFP-tagged protease and those in COS7 cells transfected with pHIV-1NL4-3/YFP contained YFP-tagged protease as examined in Western blotting. The HIV-1 virions produced were capable of infecting CD4+ MT-4 cells when the cells were exposed to the supernatant of the transfected COS7 cells (data not shown), indicating that the expressed tagged protease was enzymatically and virologically functional. In the cytoplasm of COS7 cells co-transfected with pHIV-1NL4-3/CFP and pHIV-1NL4-3/YFP, a CFP-tagged protease subunit interacts and dimerizes with a YFP-tagged protease subunit, and CFP and YFP get close because the termini are separated by only 0.5 to 1.8 nm in the dimeric form of protease (note: the representative distance was determined by analyzing the protease-DRV crystal structure (PDB ID: 2IEN)). A focused laser beam excitation of CFP (triggered by helium-cadmium laser) results in rapid energy transfer to YFP, and most of the fluorescence photons are emitted by YFP (28Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2620) Google Scholar). If the dimerization is blocked, the average distance between CFP and YFP become larger, the energy transfer rate is decreased, and the fraction of photons emitted by YFP is lowered. To help us interpret the energy transfer efficiency quantitatively, we used the acceptor photobleaching technique, in which the change in CFP emission quenching is measured by comparing the value before and after selectively photobleaching YFP, which prevents problems associated with variable expression levels. In this acceptor photobleaching approach, when FRET occurs, the fluorescence of the CFP donor increases after bleaching the YFP acceptor chromophore, which is recognized as a signature for FRET (18Sekar R.B. Periasamy A. J. Cell Biol. 2003; 160: 629-633Crossref PubMed Scopus (657) Google Scholar). Thus, the analysis of the change in CFP fluorescence intensity in the same specimen regions, before and after removal of the acceptor, directly relates the energy transfer efficiency to both donor and acceptor fluorescence. Fig. 1C illustrates representative images of co-transfected cells prior to and after YFP photobleaching, showing that, following photobleaching, YFP fluorescence of YFP-tagged wild-type protease subunit (PRWT YFP) was decreased, whereas CFP fluorescence of PR WTCFP increased. To further help us evaluate the energy transfer efficiency, we determined the ratios of cyan fluorescence intensity, determined with a confocal laser scanning microscope, after photobleaching over that before photobleaching (hereafter referred to as CFPA/B ratios). We also determined YFPA/B ratios in the same manner. If the CFPA/B ratios are >1.0, it is thought the energy transfer (or FRET) took place (18Sekar R.B. Periasamy A. J. Cell Biol. 2003; 160: 629-633Crossref PubMed Scopus (657) Google Scholar), signifying that dimerization of PR WTCFP and PRWTYFP subunits occurred. Fig. 1D shows that in the co-transfected COS7 cells (n = 23), the CFPA/B ratios were all >1.0 (CFPA/B ratios, 1.24 ± 0.11; YFPA/B ratios, 0.47 ± 0.09), demonstrating that dimerization of protease subunits occurred. Changes in Fluorescence Emission with Amino Acid Substitutions in Protease—First, it was determined whether the above-described FRET-HIV-1 assay could be used to detect the disruption of HIV-1 protease dimerization. Five amino acids at the N terminus and those at the C terminus have been shown to be critical for protease dimerization (29Babe L.M. Rose J. Craik C.S. Protein Sci. 1992; 1: 1244-1253Crossref PubMed Scopus (102) Google Scholar). As shown in Fig. 2A, two protease monomer subunits are connected by four antiparallel β-sheets involving the N and C termini of both subunits. It is of note that mature dimerized protease has as many as 12 hydrogen bonds in this N- and C-terminal region. Thus, we introduced a Pro-1 to Ala substitution (P1A), Q2A, I3A, T4A, L5A, T96A, L97A, N98A, or F99A substitution into the replication-competent HIV-1NL4-3 and found that I3A, L5A, T96A, L97A, and F99A disrupted protease dimerization, although other substitutions did not disrupt the dimerization. Several amino acid substitutions outside the N and C termini have also been known to play a role in HIV-1 protease dimerization. Ishima and Louis and their colleagues have demonstrated that the introduction of T26A, D29N, D29A, or R87K to HIV-1 protease disrupts the dimer interface contacts and destabilizes protease dimer, c