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Review 2015/04/28 Interactions of HIV-1 proteins as targets for developing anti-HIV-1 peptides Future Med. Chem.
Protein–protein interactions (PPI) are essential in every step of the HIV replication Koushik Chandra1,2, Michal cycle. Mapping the interactions between viral and host proteins is a fundamental Maes1 & Assaf Friedler*,11Institute of Chemistry, The Hebrew target for the design and development of new therapeutics. In this review, we focus University of Jerusalem, Safra Campus, on rational development of anti-HIV-1 peptides based on mapping viral–host and Givat Ram, Jerusalem 91904, Israel viral–viral protein interactions all across the HIV-1 replication cycle. We also discuss the 2Department of Chemistry, Midnapore mechanism of action, specificity and stability of these peptides, which are designed College, West Bengal, India to inhibit PPI. Some of these peptides are excellent tools to study the mechanisms of *Author for correspondence: [email protected] PPI in HIV-1 replication cycle and for the development of anti-HIV-1 drug leads that modulate PPI.
Protein–protein interactions in the
HHPID reports 15 essential HIV-1 pro- HIV-1 replication cycle
teins [25,31,41–44] (Figures 1 & 2). Three funda- Mapping the interactions between proteins mental proteins (Gag, Pol, Env) are encoded derived from host and pathogen origins is by the HIV-1 genome and they undergo pro-essential for understanding the molecu- teolysis to form the mature proteins. Four lar mechanisms of host–pathogen interac- structural proteins, matrix (MA), capsid tions [1–4]. Protein–protein interactions (CA), nucleocapsid (NC) and p6, are prod-(PPI) play a crucial role in the replication of ucts of the proteolysis of Gag. Env proteolysis HIV-1 [5–24]. HIV-1 infection results in an results in the envelope proteins gp120 and interplay between viral and host proteins or gp41 [45,46]. Pol encodes three enzymes: pro-homodimeric/oligomeric viral protein inter- tease (PR), reverse transcriptase (RT) and actions, resulting in a complex interaction integrase (IN). Encapsulated within the network between various proteins [25,26]. virus particle, the three Pol proteins play key The HIV-1-Human Protein Interaction functions in the viral replication upon infec- Database (HHPID) identified 1435 human tion. The remaining proteins (Vif, Vpr, Nef, genes encoding 1448 human proteins that Tat, Rev, Vpu) are accessory proteins [47–50]. interact with HIV-1 proteins, resulting in The database shows 43 different direct inter-2589 unique HIV-1-host protein interac- actions of HIV-1 proteins with human pro- tions [27–33]. Thirty two percent of these are teins based on activity, binding, inhibition, direct physical interactions as revealed from cleavage, complexation, modulation, deglyco-binding studies and 68% are indirect interac- sylation and upregulation. Only a part of these tions such as upregulation through activation interactions are targets for peptide inhibitors of signaling pathways. The database reveals and will be discussed here (Figure 2).
that numerous human proteins interact with more than one HIV-1 protein. Using a quan- Peptides as a tool to study PPI
titative scoring system termed mass spec- Understanding PPI requires thorough struc- trometric interaction statistics (MiST), 497 tural, biophysical and biochemical character-HIV-human PPIs involving 435 individual ization using recombinant proteins. However, human proteins and 18 viral proteins have a major hurdle is the expression and purifica- been identified [25,34–40].
tion of the interacting proteins. Some proteins 10.4155/FMC.15.46 2015 Future Science Ltd Future Med. Chem. (2015) 7(8), 1055–1077
Review Chandra, Maes & Friedler Figure 1. HIV-1 replication cycle with the essential viral proteins highlighted.
are insoluble or toxic to the expressing host, resulting present an overview of peptides derived from PPI from in low yields that hamper structural and quantitative different stages of the HIV-1 replication cycle  and studies. Using peptides for these studies provide many their implications for anti-HIV-1 drug design.
advantages relative to the recombinant proteins. Pep-tides derived from the interacting proteins enable deter- PART I: interactions between viral & host
mination of the specific interaction sites, the affinity proteins
and thermodynamic contribution (enthalpy vs entropy) Interactions between the viral capsid & host
in PPI [51–54]. Chemical synthesis of the peptides makes membrane proteins
it possible to overcome the expression and purification The initial contact between the virus and the host cell
related problems of protein production [55–57]. This is made between the viral glycoprotein gp120 (originat-
makes it technically convenient to study the PPI via ing from the Env polyprotein, PDB: 3DNL, Figure 3A)
a full-length protein and a peptide derived from the and the cell surface receptor CD4 [72,73]. CD4 is a host
complementary protein in addition to the interaction glycoprotein expressed on the surface of T helper cells,
between the two full-length proteins. Peptides derived regulatory T cells, macrophages, monocytes and den-
from binding interfaces may bind weaker than the par-
dritic cells. The binding of a highly conserved, nong- ent protein, partly due to loss of secondary structure. lycosylated region of gp120 to CD4 results in the viral Modifications such as post-translational modifications insertion into the host membrane [74–76]. Upon associa-(e.g., acetylation or phosphorylation) [58,59], labeling tion with another viral envelope protein, gp41 (PDB: (e.g., fluorescein or biotin) or incorporation of non- 2ZFC, Figure 3B), which mediates viral entry through natural amino acids can be inserted specifically into a membrane fusion, binding to CD4 occurs. This results protein sequence only using chemical peptide synthe- in a conformational change that allows gp120 to bind sis [60,61]. Peptides are an excellent model for binding to the coreceptors CCR5 or CXCR4 [77,78]; belonging studies of protein domains. Upon binding, they can to the family of G protein-coupled receptors and che-undergo conformational change mimicking the native mokine receptors [79–81]. The viral insertion causes an binding interface [62,63]. This makes peptides a useful additional conformational change in the heptad repeat tool for discovering drug leads by modulating (either regions (HR1 and HR2) of gp41 , resulting in the activating or inhibiting) PPI [64–70]. In this review, we entry of the viral capsid into the host cell via a fusion Future Med. Chem. (2015) 7(8)
future science group Interactions of HIV-1 proteins as targets for developing anti-HIV-1 peptides Review Figure 2. Selected PPI of HIV-1 proteins. (A) An interaction map between direct interactions of HIV-1-proteins (green)
with human proteins (purple). (B) An interaction map between direct interactions of viral proteins (green). Both the
interactions are involved in protein–protein interactions that served as basis for developing inhibitory peptides.
For color images please see online www.future-science.com/doi/full/10.4155/FMC.15.46
pore . This leads to the generation of epitopes for HIV-1 gp120 & CXCR4 interactionsneutralizing antibodies that prevent chemokine recep- One of the functions of gp120 is tethering of the tor binding [84,85]. Two inhibitors of fusion and entry virus to the cellular co-receptor CXCR4. CXCR4 are currently used in the clinic. Approved in 2003, the binds the bridging sheet and V3 loop of gp120 [92,93]. 36-mer peptide inhibitor T20 (Enfuvirtide) blocks a The binding between CXCR4 and gp120 involves a critical conformational change in gp41 responsible for conformational rearrangement of gp120. The soluble membrane fusion . Maraviroc is a small molecule synthetic peptide, CX4-M1, functionally mimics the antiretroviral drug, approved in 2007, which inhibits HIV-1 co-receptor CXCR4 [85,94]. The interaction the interaction between gp120 and CCR5 .
interface between gp120 and its cellular co-receptor partner CXCR4 is between the V3 loop of gp120 Peptides derived from HIV-1 Env & host proteins and the extracellular loops (ECLs) of CXCR4. The CX4-M1 peptides are derived from the ECL region The HIV inhibiting peptide database (HIPdb) reveals of CXCR4 from different HIV1 strains and bind-110 HIV inhibitory peptides that target the interac- ing was determined via direct ELISA . The bind- tions of the viral Env proteins. They aim to prevent the ing affinities between the peptides and the protein interactions between the virus and cellular cofactors were measured by surface plasmon resonance (SPR) by binding either viral envelope proteins or host pro- (Table 1C). To confirm specific binding, CX4-M1 teins [87,88]. Table 1A shows the best HIV-1 inhibitory was competed with a specific antibody, mAb447–peptide based on the prediction of antigenicity method 52D, that recognizes the V3 loop of gp120. A peptide for inhibiting Env proteins. The HIV-1 envelope pro- binding assay with CX4-M1 and V3 loop peptides tein gp41 fragment peptide (residues 568–588) is derived from the N-heptad region of gp41 Env ectodo- main . It specifically binds the phospholipid mem- gp120: HIV-1 envelope glycoprotein encoded by the brane thereby inhibiting the viral-cell fusion process. HIV env gene. The virus entry into cel s is anchored by Microcalorimetric titrations revealed that a 22-resides gp120. The process is mediated by the binding of gp120, which is exposed on the surface of the HIV envelope to tyrosine-sulfated peptide (S22 peptide) derived from specific cell surface receptors such as CD4, heparan sulfate the N-terminus of CCR5 showed a strong interac- proteoglycan. The change in the conformation of gp120 tion with the gp120-CD4 complex with K = 2.2 μM triggers fusion between the viral and host cell membranes.
(Table 1B). The process is both entropically and enthal- Integrase: Viral enzyme encoded by HIV-1, which catalyzes pically favorable. No binding was observed between the integration of the viral cDNA into the host cell genome. the gp120-CD4 complex and an identical peptide IN performs two enzymatic activities: 3′-end processing in lacking the sulfated tyrosine residues the cytoplasm and strand transfer in the nucleus.
future science group Review Chandra, Maes & Friedler Figure 3. X-ray crystal structures of some HIV-1 proteins. (A) HIV-1 gp120 trimer (PDB: 3DNL) ; (B) NTD of HIV-1
gp41 trimer (PDB: 2ZFC) ; and (C) HIV-1 RT with DNA (PDB: 3V4I) .
confirmed that the V3 loop is the crucial part of the RT is a heterodimeric protein with two asymmetric co-receptor binding site of gp120.
chains termed p51 and p66 . HIV-1 RT has three main activities: RNA-directed DNA polymerization, The viral enzyme reverse transcriptase
DNA-directed RNA polymerization and exonuclease HIV-1 reverse transcriptase (RT) produces a viral cDNA via degradation of RNA . RT is the target of numer-based on the viral RNA (PDB: 3V4I, Figure 3C). The ous small molecule antiretroviral drugs used in the DNA is later integrated into the host cell genome . clinic . AZT is a Nucleoside analog RT Inhibitor Future Med. Chem. (2015) 7(8)
future science group Interactions of HIV-1 proteins as targets for developing anti-HIV-1 peptides Review Table 1. Peptides that inhibit viral entry.
(A) Env and CD4 interaction
(B) gp120 and CCR5 interaction
MDYQVSSPIY(SO -)DINY(SO -)YTSEPSQK (C) gp120 and CXCR4 interaction (ECL1)CX4-M1
PPI: Protein–protein interaction.
(NRTI) that acts as a chain terminator of growing viral cDNA into the host genome (Figure 1) [111–115]. It DNA strand and was approved as an anti-HIV drug has three functional domains responsible for integra-in 1987. In 1996, Nevirapine was approved as the first tion process: the N-terminal domain (NTD), the cata-non-nucleoside RT inhibitor (NNRTI) that inhibits lytic core domain (CCD) and the C-terminal domain the RT polymerization activity.
(CTD) [116,117]. IN has two enzymatic activities: first, 3′-end processing in the cytoplasm [111,118] in which two The HIV-1 RT & A3G interaction IN dimers  bind the long terminal repeats (LTR) of During reverse transcription, the human cytidine deam- the viral DNA and remove a pGT dinucleotide from the inase APOBEC3G (A3G) eliminates HIV-1 infection 3′-end of each strand. After nuclear transport [120,121], by inducing deamination of the cytosine residues to the strand transfer reaction is carried out by an IN tet-uracil in the negative viral DNA strand [100–108]. Using ramer [122–124] resulting in integration of the viral DNA a cell-based co-immunoprecipitation (coIP) assay, the into the host genome. Finally, the single-stranded gaps direct interaction of A3G with RT was detected both in transfected cells and in the produced viruses. No other viral components are needed for this interaction.
Deletion analysis with a series of T7-tagged RT-dele-
tion mutants (T7-RT1–243, T7-RT1–323 and T7-RT1–439)
determined that the RT-binding domain is located
at the N-terminal region of A3G65–132 [101,109,110]. The
polypeptide A3G65–132 inhibited the interaction between
A3G and the viral RT (PDB: 3VOW, Figure 4A,
Table 2A) . The RT-binding polypeptide inhibited
the anti-HIV effect of A3G on RT. Competitive coIP Figure 4. Peptides derived from human APOBEC3G.
in cells co-expressing both RT and A3G using sev- (A) Crystal structure of human A3G. The RT-binding
eral A3G derived polypeptides showed that A3G65–132 A3G 65–132 peptide is shown in cyan (PDB:3VOW) , (B) Crystal structure of APOBEC3G catalytic core
significantly disrupted the A3G-RT binding.
domain (CCD); the Vif-interactions regions are: A3G 211–225 (magenta), A3G 263–278 (cyan), A3G 331–345 Interactions of the HIV-1 integrase
(green) and A3G 353–367 (red) (PDB:3IR2) . HIV-1 integrase (IN) plays one of the key roles in the viral For color images please see online www.future-science.
replication cycle by integrating the reverse transcribed com/doi/full/10.4155/FMC.15.46 future science group Review Chandra, Maes & Friedler Table 2. Peptides that inhibit the viral enzymes reverse transcriptase and integrase.
(A) RT & A3G
(B) IN dimerization IN 93–107(INH1)
IN 129–139(NL9) IN 129–139W131A IN 171–187(α5) IN 196–210(α6) IN 196–206(α6S) IN 151–175(K156 E (B) Cellular partner LEDGF/p75 354–378
LEDGF/p75 355–377 LEDGF/p75 361–370 LEDGF/p75 362–369 LEDGF/p75 402–413 (C) Phage display
†Inverted sequence with d-amino acids.
PPI: Protein–protein interaction.
between the viral DNA and target DNA are repaired by tors. Some of the peptides (IN 95–109, IN 97–108, IN 171–187 the host DNA repair machinery [125–127]. The equilib- and IN 196–210) showed very mild IC for both 3′-pro- rium between dimeric and tetrameric IN is of extreme cessing and strand transfer in vitro. IN 147–175, which is importance in the integration process, making it an derived from IN, inhibited IN at 600 μM concentration attractive target for drug design . In 2007, Ralte- by partly blocking the active site. The peptide inhib- gravir was the first IN inhibitor approved for clinical ited the catalytic activity of IN by binding it through use. Another IN inhibitor, Elvitegravir, was approved a protein-peptide coiled-coil structure [130,133–135]. Two for clinical use in 2012 [128,129]. Both inhibitors block peptides derived from the α1 and α5 helices of the IN by binding directly to the IN-DNA complex formed CCD, (INH1 and INH5) specifically bound to the during the integration of the viral DNA into the host dimerization interface of the CCD of IN . The cell genome [128,129].
IC for 3′- processing by INH1 was 250 μM and by INH5 was 11 μM. By inhibiting the 3′-endonuclase Peptides derived from the dimerization activity of IN with IC values in the low micromolar range, three peptides (α1, α5, α6) also inhibited the IN The dimerization interface of IN is an excellent start- dimerization . The truncated peptide (NL6–5) and ing point for peptides that would inhibit dimer forma- retro-inverso-peptides (RDNL6, RDNL9) retained the tion [130–132]. Several peptides have been designed (PDB: inhibitory activity by disrupting the IN dimer and tet-3L3U, Figure 5A, Table 2B) but due to relatively low ramer formation [138,139] All the peptides were derived binding affinity to IN, they did not succeed in disrupt- from the CCD of IN, which is the only domain that ing the dimeric IN and hence were not efficient inhibi- mediates IN dimerization (Figure 5A) [138,139].
Future Med. Chem. (2015) 7(8)
future science group Interactions of HIV-1 proteins as targets for developing anti-HIV-1 peptides Review Figure 5. Peptides derived from domains of HIV-1 integrase. (A) Crystal structure of HIV-1 IN catalytic core domain
(CCD, beige and green) dimer illustrates the important regions of the IN-dimerization interface from where
peptides were derived: IN 93–107 (INH1, α1, NL6) (magenta), IN 171–208 (α5, α6, α6s) (magenta) (PDB:3L3U) .
(B) Crystal structure of dimeric IN CCD and LEDGF/p75 IBD (gray) showing interacting regions: LEDGF/p75 354–378
(cyan), LEDGF/p75 361–370 (red), LEDGF/p75 402–411 (magenta) .
For color images please see online www.future-science.com/doi/full/10.4155/FMC.15.46
Peptides derived from cellular proteins that LEDGF/p75402–413 are important for optimal binding and inhibition of IN (Figure 5B) [69,153–156]. A library of Targeting host proteins is risky since it may affect cell cyclic peptides (CPs) derived from LEDGF/p75361–370 viability and produce undesired toxicity. Therefore, the was screened for in vitro IN binding and inhibition . best strategy is to study the interactions between IN and One of these peptides, c(MZ4–1) was a potent and sta-host proteins by finding peptides derived from the IN- ble inhibitor of IN in vitro. NMR and docking studies binding region of the cellular proteins. These peptides revealed that c(MZ4–1) possessed a conformation almost will potentially bind the viral protein and inhibit the identical to the parent IN-binding loop from the IBD of interaction with less potential for undesired side effects.
LEDGF/p75. An AlphaScreen assay with these peptides also accounted for IN-LEDGF/p75 interaction .
The IN-LEDGF/p75 interaction A random peptide phage display strategy was In addition to binding the viral DNA [140–142], IN inter- adopted to identify a linear peptide, LEDGF/p75325– acts strongly with the cellular transcriptional co-factor 530, that bound specifically to the IBD of LEDGF/p75. LEDGF/p75 . LEDGF/p75 tethers the IN-DNA Based on this, small CPs (CP64 and CP65) inhibitors complex to the host chromatin, where the final integra- of the IN–LEDGF/p75 interaction (IC for CP64 tion steps take place [140,144–151]. The IN-LEDGF/p75 is 35.88 μM and IC for CP65 is 59.89 μM) were is a crucial interaction in the replication cycle, making developed. These peptides inhibited HIV replica-it as a fundamental target for anti-HIV drug design.
tion in different cell lines without displaying toxicity The structure of IN CCD in complex with the (Table 2C) . Saturation transfer difference (STD) LEDGF/p75 IN binding domain (IBD) shows a pseudo two-fold symmetry where an IN CCD dimer Key termsbinds two LEDGF/p75 IBD at either side (PDB: 2BJ4, Alanine scan: Screening technique for determining of Figure 5B) . The IBD interacts with IN via two loops. the contribution of specific residues to the function and Our lab rationally designed peptides based on these interactions of a protein. Each residue is sequential y replaced by alanine and the function/interaction of the loops and shorter variants (LEDGF/p75361–370, LEDGF/ mutant peptide/protein is compared with the parent p75402–411) . All of these bound IN with micromolar peptide. Loss of function/interaction means that the affinities and inhibited the in vitro enzymatic activities original residue was important for the binding/activity. Alanine is used since it is the simplest chiral residue and both in presence and absence of LEDGF/p75 (Table 2B). thus mimic a loss of a side chain without a conformational In addition, these peptides inhibited the integration of change or an introduction of a new function.
viral cDNA and HIV-1 replication in infected cells, by Cyclic peptides: Cyclization improves the pharmacological shifting the IN oligomerization equilibrium toward a properties of peptides. They are conformational y rigid, stable tetramer in the cytosol. Further studies including resistant to protease degradation and in many cases have homology modeling, alanine scan and NMR analysis improved affinity and specificity as well as cell penetration revealed that all the residues of LEDGF/p75361–370 and properties.
future science group Review Chandra, Maes & Friedler NMR confirmed that the residues in CP64 strongly prevent PR-mediated cleavage at specific Gag sites and bound to LEDGF/p75 and not to HIV-1 IN.
also binds CA to prevent core formation .
Stapled peptides that target IN-mediated The interaction between Gag p6 & human integration & the IN-LEDGF/p75 interaction Two-domain crystal structures of IN show that the HIV-1 p6 is a Gag cleavage product that plays an two monomers of dimeric IN are tethered via strong important role in regulating capsid processing, facili-helix-helix (α1:α5′ and α5:α1′) interactions [159,160]. tating virus budding and incorporation of the viral Using the ‘sequence-walking' strategy, two potent IN accessory protein R (Vpr) into virions. These pro-inhibitors termed NL6 and NL9  were revealed. cesses require interactions between the human tumor NL6 has an α-helical structure and is part of the α1 susceptibility gene 101 (Tsg101) protein and the CTD helical domain. A series of hydrocarbon stapled pep- of p6 . Tsg101 is a part of the endosomal sorting tides derived from NL6 (NLH2-NLH16, NLX1, complex required for transport-I (ESCRT-I), which NLX2) enhanced interfacial interaction and cell- assists the ubiquitylation of Gag and facilitates viral permeability compared with the parent NL6 peptide assembly and budding [177–180]. Successful HIV-1 bud-through stabilization of the α1 domain  as con- ding requires an interaction between the tetrapeptide firmed by CD studies. Increasing the α-helical content PTAP, derived from residues 3–6 in p6, with the also increased the IN inhibitory activity at the 3′-pro- ubiquitin E2 variant (UEV) domain of Tsg101 (PDB: cessing step, inhibition of the strand transfer reaction 3OBU, 3OBX, Figure 6). Blocking this interaction and the IN-LEDGF/p75 interaction, cytoprotective inhibits virion formation [181–183].
activity (EC ), cell death activity (CC ) and thera- Peptides containing the PTAP motif are potential peutic index (ratio of CC to EC ). Combining pairs inhibitors of the interaction between Gag p6 and of α-helical peptides effectively inhibited IN catalytic Tsg101. A peptide derived from p65–13 bound Tsg101 activities. The most active pair was unstapled NLH5 (Table 3A) . NMR studies showed that the peptide and stapled NLH6 (IC values of 9 ±1 μM for 3'-pro- bound to Tsg101 in a groove that interacts with the cessing and 6 ±1 μM for strand transfer ). The PTAP residues with K = 3 μM [177,183]. The struc- pairs were designed with a covalent hydrocarbon staple ture showed that binding of E2 ubiquitin-conjugating spanning i and i + 4 residues that did not show inhibi- enzymes to UEV domain of Tsg101 was hampered tion in the alanine scan [163,164]. Most of the stapled upon PTAP binding (Figure 6). Structure activity rela-peptide pairs inhibit the IN-LEDGF/p75 interaction. tionship (SAR) studies of this peptide, which included Six peptides (NLH2, NLH3, NLH5, NLH6, NLH15, conversion to P3 polycyclic oxime derivatives in the NLH16) inhibited HIV-1 replication in MT-4 cells. PTAP domain, improved binding to Tsg101 by 15- to Fluorescein-tagged NLH6 (termed NLX-1) pen- etrated cells and inhibited the target IN. MT-4 cells To develop effective competitive inhibitors, a tech- showed significant cellular uptake of NLX-1, which nique for genetically selecting CPs that inhibit specifi-was localized mainly to the cytoplasm with minimum cally the p6 Gag-Tsg101 interaction was used . This distribution to the nucleus. The cell-permeability and technique, called SICLOPPS (split intein-mediated cir-enhanced potency of the stapled peptides makes them cular ligation of peptides and proteins), allowed iden-lead IN inhibitors.
tification of new CPs that specifically blocked the p6 Gag-Tsg101 interaction and consequently inhibited HIV Mapping HIV-1 Gag & host cellular proteins
replication. After several rounds of screening, the selected CPs had no resemblance to the original sequence of the HIV-1 Gag is a viral polyprotein expressed during the interacting sites in either p6 Gag or Tsg101. Of these, late phase of the replication cycle. Cleavage of Gag by the CP11 inhibited the formation of virus-like particles viral PR produces the structural proteins of the mature (VLP) in cultured cells with IC of 7μM and showed virion: the matrix (MA), capsid (CA) and nucleocapsid better stability compared with the linear p65–13.
(NC) proteins. MA is the N-terminus of Gag, followed by a CTD termed p6 and two spacer regions that sepa- The p6 Gag & cyclophilin interaction rate CA from NC and NC from p6. The Gag products Another partner of p6 Gag is the cellular cyclophilin take part in viral self-assembly and release of virions A (CypA) protein, which acts as a prolyl isomerase from the infected cells, thus making it critical for viral (PPIases). CypA also acts as a molecular chaperone particle morphogenesis and replication within the liv- and assists protein folding, assembly and transporta- ing cells [165–174]. The maturation inhibitor Bevirimat is tion processes. CypA is incorporated into newly bud-currently in clinical trials. It targets the Gag protein to ding particles of HIV-1 and thus can be considered as Future Med. Chem. (2015) 7(8)
future science group Interactions of HIV-1 proteins as targets for developing anti-HIV-1 peptides Review a key target in future antiretroviral therapy . p6 contains a relatively high content of proline residues, at positions 5, 7, 10, 11, 24, 30, 37 and 49. Proline cis/trans isomerism was observed for all these proline resi-dues and more than 40% of all p6 Gag proteins show at least one proline in cis-orientation. 2D proton NMR of full length p6 Gag or p6 Gag-derived peptides with CypA revealed that it interacts with all proline residues of p6 Gag through a prolyl-peptidyl cis-trans isomerase (PPIase).
The modulation of HIV-1 p6 function by CypA was explored by the synthesis of full length p6 and several Figure 6. Peptides derived from Tsg101 UEV. (A) Crystal
p6 fragments (p61–14, p61–21, p623–32, p632–42, p643–52 structure of the Tsg101 UEV domain (light brown) in and p623–52) and by using NMR and Surface Plasmon complex with a HIV-1 Gag P7A mutant p6 5–13 PSAP Resonance (SPR) (Table 3A) . Catalytic amount peptide (green) (PDB:30BX) . (B) Crystal structure
of CypA is sufficient to interact with all the proline of the Tsg101 UEV domain (blue) in complex with a residues of p61–52 (molar ratio 1: 283; Table 3A) and HIV-1 PTAP (5–13) peptide (red) (PDB:30BU) . For color images please see online www.future-science.
hence PPIases activity in vitro. However, there was low com/doi/full/10.4155/FMC.15.46 affinity binding of CypA to p6 fragments compared with binding to full-length p6. Another important peptides have a higher resistance to degradation by inhibitor of CypA is cyclosporine A which was found proteases (Table 3B) [197,198,201–205].
to suppress both the production and the release of new virions [189,190].
The Tat-p53 interactionThe cellular tumor suppressor p53 is a homotetra- Interactions of HIV-1 Tat
meric transcription factor that induces cell cycle The HIV-1 trans-activator of transcription (Tat) arrest or apoptosis upon oncogenic stress . NMR protein is a small viral auxiliary protein that con- and x-ray crystallography revealed that the p53 tet- tains 101 or 86 residues, depending on the HIV ramerization domain (p53 Tet; residues 326–355) strain [191,192]. The Tat protein can be divided into six has a dimer of dimers structure [207,208]. Depending regions: an acidic region (residues 2–11), a cysteine- on its concentration, p53 Tet exists in equilibrium rich domain (residues 22–37), the hydrophobic core among different oligomeric forms [209,210]. p53 inhib-(residues 38–46), a basic region (residues 47–57), its Tat-mediated LTR transcription . The viral the glutamine-rich domain (residues 58–72) and Tat binds p53 Tet as was shown by yeast two-hybrid the RGD motif (residues 72–86) [193,194]. The basic system [209,212]. The CTD of p53 (residues 341–355) region of Tat binds to the negatively charged mRNA interacts specifically with the Tat residues 49–57 in in the Tat-activation region (TAR) [195,196]. The bind- the arginine-rich motif (ARM) . Tat 73–86 can ing of Tat to TAR promotes a prolongation of the bind p53 with the assistance of cellular proteins such transcription due to conformational change of the as NF-κB and CBP/p300, as observed by in vivo TAR during binding of host cell kinases that phos- phorylate the RNA polymerase II complex. The six To quantitatively understand the molecular basis of Arginine residues in Tat47–57 are crucial for Tat-TAR Tat-p53 interaction during HIV-1 replication cycle, recognition [197–200].
our laboratory synthesized Tat-derived peptides (Tat1– The peptide Tat47–57 specifically disrupted the TAR- 35 and Tat47–57) and studied their binding to the p53 RNA recognition by blocking the production of viral tetramerization domain (Table 3C) . The binding transcript and also interrupted the formation of two between p53 Tet and Tat47–57 is purely cooperative and cellular cofactors, cyclin T1 and its cognate kinase is temperature-dependent. NMR studies revealed that CDK9, responsible for transcriptional elongation E343 and E349 from p53 Tet are the major Tat47–57 from the viral long terminal repeat (LTR) [197,198,201– binding residues. The binding mechanism involves 205]. Increasing the number of Arginine residues on electrostatic interactions .
the hairpin scaffold of Tat-derived peptides dra-matically decreased the specificity for binding the The interaction of HIV-Vif with the host TAR-RNA. In contrast, fewer Arginine residues in cellular protein APOBEC3Ga Tat-derived peptide of the same length increased The HIV-1 virion infectivity factor (Vif) is required the TAR-RNA binding specificity. Arginine-rich Tat for the virus replication [215,216]. Vif counteracts A3G future science group Review Chandra, Maes & Friedler by targeting it for proteosomal degradation and by interaction between Vif and A3G and thus their inhi-direct inhibition of its enzymatic activity (PDB: bition may rescue the antiviral activity of A3G and 3IR2, Figure 4B) . Both activities involve a direct inhibit HIV-1 propagation [218–220]. Vif binding to Table 3. Peptides derived from interactions between viral (Gag, Tat, Vif and Vpr) and host proteins.
(A) Interaction between MA and TCR
Gag p6 and Tsg101 interaction p6 5–13 PSAP peptides
p6 Gag and CypA interaction p6-UEV interaction (B) Interactions of Tat with host proteins
Tat derived peptides
(C) The Tat-p53 interaction
Tat derived peptides
P53 derived peptides p53 326–355 R342A p53 326–355 L344P p53 326–355 L344A p53 326–355 E346A †AghTat is (S)-2-Amino-6-guanidinohexanoic acid and AgbTat is (S)-2-Amino-4-guanidinobutyric acid. These are non natural amino acids.
PPI: Protein–protein interaction.
Future Med. Chem. (2015) 7(8)
future science group Interactions of HIV-1 proteins as targets for developing anti-HIV-1 peptides Review Table 3. Peptides derived from interactions between viral (Gag, Tat, Vif and Vpr) and host proteins (cont.).
(D) Interactions between Vif and host proteins
Vif and A3G interaction
Vif and Cullin5 interaction (E) Interactions of Vpr
Vpr and CypA interaction
Vpr 75–90 (R80A) Vpr 75–90 (R76Q V83I Vpr 75–90 (R76Q V83I †AghTat is (S)-2-Amino-6-guanidinohexanoic acid and AgbTat is (S)-2-Amino-4-guanidinobutyric acid. These are non natural amino acids.
PPI: Protein–protein interaction.
A3G results in polyubquitination and degradation of from three distinct regions in Vif: residues 8–45 from A3G by forming a E3 ubiquitin ligase complex consist- the NTD, residues 154–192 from the CTD contain- ing of ElonginB and C, Cullin5 and RING finger pro- ing the conserved motif 161PPLP164 and a central region tein 1 [221,222]. The mutation K128D in A3G abrogated between residues 83–99 [230,231]. The A3G-derived pep-the interaction with Vif [223,224].
tides A3G143–157, A3G211–235 and A3G263–277 bound full- The peptides Vif14–17, Vif 22–26, Vif40–44 and Vif69–72 length Vif and Vif-CTD. The peptide array experiment inhibited the A3G-Vif interactions (Table 3D) . revealed that peptides A3G 31–52, A3G 166–180, A3G 211–225,
Deletion mutagenesis of A3G also showed that A3G 263–277 and A3G 331–367 also bound to Vif.
Vif54–124 and Vif105–156 peptides are critical for the
interaction . An in vitro Vif-A3G binding assay The interactions of Vpr with host cellular
between GST-tagged Vif and His-tagged A3G and proteins
a Fluorescence Resonance Energy Transfer (FRET) The viral protein R (Vpr) is the only virion associated reg-
assay between GST-Vif and biotinylated A3G110–148 ulatory protein and is not a component of the virus poly-
confirmed their interactions (Table 3D) .
protein precursors. It assists the nuclear import of the pre- Mapping the Vif-A3G interaction by using peptide integration complex (PIC) in nondividing host cells . arrays resulted in defining the precise binding interface Vpr is crucial for effective HIV-1 infection of target (Table 3D) [226–229]. A3G bound nine Vif-derived peptides CD4+ T cells and macrophages [233–235]. Vpr interacts future science group Review Chandra, Maes & Friedler with numerous cellular proteins in order to perform its adenine nucleotide translocator (ANT) [246,247]. The nuclear import and G cell cycle arrest functions.
VDAC and ANT interaction is based on permeability transition pore (PTP) as a result of dynamic multiprotein Interaction between Vpr & CypA complex formation at inner and outer mitochondrial One of the key Vpr interacting protein is cyclophilin A membrane contact sites.
(CypA) . Cis-trans prolyl isomerization of the highly A TEAM-VP (Targeted to Endothelial Apoptogenic conserved proline residues in Vpr, such as Pro5, Pro10, Mitochondrio-active Vpr-derived Peptide) peptide was Pro14 and Pro35, is catalyzed by CypA. SPR experiments designed based on α β binding and endothelial apop- showed that the heptapeptide CypA 32–38(32RHF- togenic sequences derived from the mitochondria active PRIW38) mediates the binding between CypA and the portion of Vpr. TEAM-VP peptide is combined with a N-terminal region of Vpr . P35A mutation disrupted tumor blood vessel RGD-like ‘homing' motif and a mito-the Vpr-CypA interaction. In the mutant peptide Vpr75–90 chondrial membranes permealization (MMP)-inducing (R80A), the replacement in the C-terminal region of Vpr sequence. It is composed of the cysteine mediated CP hampered the co-IP of Vpr with CypA [238,239].
sequence GGCRGDMFGC and a Vpr67–82 sequence The above observations together with the significant derivative (Table 3E). The cyclic core ‘GGCRGDMFGC' amount of CypA in the virion  led to the design of of TEAM-VP specifically bound to VDAC and ANT Vpr-based peptides to study the Vpr - CypA interaction and internalized into α β -expessing cells through its (Table 3E) [241,242]. SPR and ITC studies revealed the cyclic-RGD motif. .
strong binding affinities of C-terminal Vpr75–90 (K = 0.28 μM) and N-terminal Vpr30–40 (K = 1 μM) peptides. PART II: interactions between viral proteins
Other C-terminal Vpr peptides such as Vpr69–78, Vpr75–84, The Env–MA interactionVpr81–90 and Vpr87–96 interacted weakly with CypA. The The matrix protein p17 (MA) originates from the weakest binding response was observed for mutant pep- Gag precursor protein, p55gag . It is N-terminally tides such as Vpr75–90 R80A (K = 7.5 μM) and Vpr75–90 myristylated and binds to the viral inner membrane or R76Q, V83I, R80A, T841 (K = 4.7 μM) as compared the inner leaflet of the plasma membrane (PM) of the with the wild type peptide. NMR studies revealed that infected cells . MA is involved in nuclear import of the mutations did not influence the secondary structure the viral DNA . A specific interaction between p17 of the C-terminal binding domain of Vpr.
and Env was revealed by the co-expression of Env pro-teins that influenced the assembly of Gag particles. The The interaction between Vpr & the WXXF motif membrane-proximal amino terminus of p17 in the Gag of host cell proteins precursor closely associates with the membrane in the The conserved WXXF motif of uracil-DNA-glycosylase mature particle indicating that p17 participates in the mediates the intracellular binding of Vpr with uracil specific Env incorporation into the viral particles .
DNA glycosylase. Many WXXF-including peptides Several p17 peptides (p17l-12, p1712–29, p1730–52, p1753– have domain-specific interactions with Vpr. The fusion 87, p1787–115 and p17115–132), derived from all the six parts of the WXXF dimer to the chloramphenicol acetyl trans- of p17, were synthesized (Table 4A) [253,254]. The anti-ferase (CAT) gene demonstrated that the WXXF dimer- genic epitopes was examined for anti-HIV-1 p17 anti- CAT construct induced CAT activity inside the virions body (p17 Ab) in the serum of an HIV1 carrier. p17l-12, through Vpr-dependent docking .
p1712–29, p1730–52 were highly recognized in the serum Phage display peptide screening predicted that more and led to inhibition of virus multiplication as tested than 90% peptides having consensus motif WXXF effi- using ELISA. The purified antibodies obtained from the ciently binds Vpr protein [243,244]. Similarly, Vpr binding patient using the p17-derivated peptide immunoaffinity peptides from GST-Vpr panning also revealed a WXXF columns confirmed that the reactivity of p1730–52Ab to consensus motif . Nine peptides were found to bind p17 was the highest among the antibodies.
Vpr (Table 3E) .
The Gag–PR interaction The Vpr interaction with cell-surface α β in PR cleaves the Gag and Gag-Pol precursors into active endothelial cells viral proteins such as p1gag, p2gag, p6gag, p7gag, p17gag and Vpr targets mitochondrial membranes to trigger apopto- p24gag [45,255–257]. The cleavage of the Gag precursors is sis and cell death. The internalization of cyclic RGD in necessary for maturation and HIV-1 infectivity. p2gag is endothelial cells for cellular apoptosis is mediated by the an inherent suicidal inhibitor of PR due to its strong in cell surface receptors α β integrins. The Vpr induced vitro inhibition of the proteolytic cleavage of the recom- apoptotic cell death involves the interactions of Vpr with binant Gag precursor into functional structural units the voltage-dependent anion channel (VDAC) and the (p17gag and p24gag) . After the viral maturation, p2gag Future Med. Chem. (2015) 7(8)
future science group Interactions of HIV-1 proteins as targets for developing anti-HIV-1 peptides Review inhibits PR activity in released viral particles and thus essential for PR binding and blocking proteolysis blocks the autolysis of HIV-1 virions.
(Table 4B). Vif21–65 inhibited PR five times better than PR is one of the most common anti-HIV drug tar- full-length Vif. Vif-derived peptides such as Vif30–65 and gets and many FDA approved anti-HIV drugs are PR Vif78–98 specifically inhibited the Vif-PR interaction in inhibitors [86,97]. The nonapeptide (AEAMSQVTN) vitro and blocked the production of viruses in HIV-1-in-derived from the N-terminus of p2gag inhibited HIV-1 fected cells [262,263]. Vif88–98 inhibited PR dimerization. PR activity in vitro to prevent autolysis of the virion after Two PR-derived peptides PR1–9 and PR94–99 abrogated sequential processing and reorganization of the virion Vif function as an A3G neutralizer and inhibited Vif-PR core (Table 4B) . Further SAR studies with p2gag binding in a dose-dependent manner . This means revealed that alanine replacements (M4A and T8A) and that PR1–9 competed with PR for the Vif binding site.
deletion of Asn9 from the nonamer (AEAMSQVTN) decreased the PR inhibitory properties. However, the Vpr interactions with RT & INother mutated peptides did not have inhibitory activity.
RT, IN and Vpr are in close spatial proximity within the PIC, allowing them to interact with each other . The Vif–PR interaction The interaction between RT and IN involves the single- Vif blocks the cleavage activity by directly interacting stranded viral RNA copied into integration-competent with PR. Vif stably blocks the premature activation of double-stranded DNA by RT, DNA polymerase and PR in cytoplasm, which is circumvented during particle ribonuclease H (RNaseH). Then the PIC is imported assembly . The NTD of Vif (residues 1–96) inhibits to nucleus by IN and Vpr for integration . RT and the PR cleavage in vitro and in bacteria. Both Vif and PR IN physically interact with each other and the full-are present in the mature virions . Vif regulates PR in length Vpr and its isolated CTD can interfere with the the virion at the early stage of infection . Several Vif- IN-mediated integration activity in vitro .
derived peptides inhibited PR-mediated cleavage of Gag A library of Vpr-derived peptides was screened for in vitro and during viral protein expression in peripheral their ability to bind directly to RT and IN in vitro and blood lymphocytes . Vif1–38 and Vif 1–65 and Vif10–96 to inhibit their enzymatic activities (Table 4B) [265–267]. peptides were highly stable toward proteolysis. Vif21–65 is Dot-blot binding assay showed that the C-terminal Vpr Table 4. Peptides derived from interactions between viral proteins.
(A) Interaction between Env and MA
Env and MA
(B) Interactions of Protease
Protease and Gag
p2 gag pep# mutant1 p2 gag pep# mutant2 p2 gag pep# mutant3 p2 gag pep# mutant4 p2 gag pep# mutant5 PPI: Protein–protein interaction.
future science group Review Chandra, Maes & Friedler peptides (Vpr57–71 and Vpr61–75) efficiently bound RT and Fuzeon® (Enfuvirtide) was approved for clinical use IN. Molecular docking of Vpr57–71 into the 3D structure against HIV [84–86,268,269]. Current research in anti-of RT and of the two peptides Vpr33–47 and Vpr61–75 into HIV drug design is focused on stabilizing lead peptides the IN CCD were carried out to understand the bio- using different strategies such as cyclization, peptoids chemical effects such as steric hindrance and conforma- and more [268–273].
tional changes of the active sites. DNA polymerase as well Peptides serve as excellent starting points for the as RNase H activities of RT were significantly inhibited design of peptidomimetics and the development of new by Vpr57–71, Vpr65–79 and Vpr69–83 with IC values in the small molecule drug leads based on their sequences and range of 0.22–2 μM. DNA primer extension by RT was conformations. Currently, many of the FDA-approved also inhibited by Vpr53–67, Vpr57–71, Vpr61–75, Vpr65–79 and anti-HIV drugs in the clinic, such as Indinavir, Ritona-Vpr69–83. Vpr33–47, Vpr57–71, Vpr61–75 and Vpr65–79 were able vir, Saquinavir and Lopinavir are the result of gradual to abrogate IN strand transfer activity. The three peptides conversion from a peptide to a small molecule [269,274–Vpr57–71, Vpr61–75 and Vpr65–79 inhibited the 3'-end process- 278]. These small molecules are mostly peptidomimetic ing activity of IN whereas the disintegration was blocked hydroxyethylene or hydroxymethylamine HIV-1 pro-by Vpr33–47, Vpr69–83, Vpr57–71, Vpr61–75 and Vpr65–79.
tease inhibitors. Other types of small molecules such as ADS-J1, ADS-J2, XTT formazan, NB-2, NB-64, Conclusion & future perspective
AOP-RANTES, PSC-RANTES, Vicriviroc, Mara- In this review, we described the PPI in the HIV-1 rep- viroc and Aplaviroc are also the outcome of peptido- lication cycle that are targets for inhibition by peptides mimetic approaches. They act by targeting the HIV-1 and from which inhibitory peptides were derived. These entry through gp120, gp41, CCR5 and CXCR4. This PPI include both viral–cellular and viral–viral protein approach may be used in the future for other PPI as interactions. Most of the peptides reported are derived described above.
from viral–host PPI and not from viral–viral PPI, indi-cating that the host–viral interactions are more promis- Financial & competing interests disclosure ing drug targets. Current research is focused on devel- A Friedler is supported by a grant from the Israeli Science oping peptides libraries based on in vitro and in vivo Foundation (ISF) and by the Minerva Centre for Bio-Hybrid experiments that will be later modified into small mol- Complex Systems. The authors have no other relevant affilia- ecule inhibitor. The peptides are discovered using dif- tions or financial involvement with any organization or entity ferent approaches, and different assays were performed with a financial interest in or financial conflict with the subject to analyze their quantitative or qualitative binding to matter or materials discussed in the manuscript apart from viral proteins and their effect on HIV-1 infectivity.
Peptides do not serve only as tools for studying No writing assistance was utilized in the production of this PPI, but have clinical use against HIV. The peptide, manuscript.
Executive summary • Protein–protein interactions (PPI) are essential in every step of the human immunodeficiency virus (HIV) replication cycle.
• Mapping the interactions between viral and host proteins, as well as between the viral proteins themselves, is a fundamental target for the design and development of new therapeutics.
• Peptides are excellent tools to study the mechanisms of PPI in HIV-1 replication cycle and for the development of anti-HIV-1 drug leads that modulate PPI.
• These peptides can be later developed into small molecules, which can be used as drugs.
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future science group
Race and Medicine Genetic studies of population differences, although controversial, promise David Goldstein of University College in clues to disease as well as new drug targets, scientists believe London agrees: "If you say on average the difference between West Africans and Eu- Mention race and medicine in a group of racial identity biologically irrelevant. But ropeans is slight, that does not rule out a