The retroviral RNA genome encodes for three enzymes essential for viral replication: HIV-1 protease PR, HIV-1 reverse transcrip-tase RT and HIV-1 integrase IN.. Peptide-based inhibitors
Trang 1R E V I E W A R T I C L E
Modulation of the oligomeric structures of HIV-1 retroviral enzymes
by synthetic peptides and small molecules
Nicolas Sluis-Cremer1and Gilda Tachedjian2
1
Department of Medicine, Division of Infectious Diseases, University of Pittsburgh, PA, USA;2AIDS Molecular Biology Unit, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia
The efficacy of antiretroviral agents approved for the
treat-ment of HIV-1 infection is limited by the virus’s ability to
develop resistance As such there is an urgent need for new
ways of thinking about anti-HIV drug development, and
accordingly novel viral and cellular targets critical to HIV-1
replication need to be explored and exploited The retroviral
RNA genome encodes for three enzymes essential for viral
replication: HIV-1 protease (PR), HIV-1 reverse
transcrip-tase (RT) and HIV-1 integrase (IN) The enzymatic
func-tioning of each of these enzymes is entirely dependent on
their oligomeric structures, suggesting that inhibition of
subunit-subunit assembly or modulation of their quaternary structures provide alternative targets for HIV-1 inhibition This review discusses the recent advances in the design and/or identification of synthetic peptides and small mole-cules that specifically target the subunit–subunit interfaces of these retroviral enzymes, resulting in the inactivation of their enzymatic functioning
Keywords: protease; reverse transcriptase; integrase; oligo-meric structure; inhibiting protein–protein interactions
In 1983 HIV was identified as the etiologic agent of AIDS
[1,2] During the past 18 years a tremendous effort has been
placed in the identification and/or development of
com-pounds that effectively attenuate HIV-1 infection To date,
16 anti-HIV agents have been approved by the United
States FDA for administration to HIV-1 infected
individ-uals These antiviral agents target the active sites of two
retroviral enzymes, protease (PR) and reverse transcriptase
(RT), and can be further divided into three different
therapeutic classes; PR active site inhibitors, nucleoside (and
nucleotide) reverse transcriptase inhibitors (NRTI) and
non-nucleoside reverse transcriptase inhibitors (NNRTI)
However, due to the long-lived nature of the HIV-1
infection as well as the genetic plasticity inherent to the
virus, emergence of viral resistance to these antiretroviral
agents is inevitable Furthermore, as many of the
com-pounds from the same therapeutic class exhibit similar chemical structures and mechanisms of action, the emer-gence of viral resistance to one drug frequently results in cross-resistance to other compounds Thus, the identifica-tion of addiidentifica-tional viral targets and the development of new classes of antiviral compounds are essential in the fight against HIV/AIDS In this regard, many promising com-pounds have been identified that target different steps in the HIV-1 viral life cycle including viral entry and fusion, proviral DNA integration as well as viral assembly (for reviews see [3,4])
Physical interactions between proteins play a critical role in many biological processes including signal trans-duction, cell cycle and gene regulation, and viral assembly and replication [5–7] Furthermore, many protein–protein interactions provide therapeutically worthwhile targets In this regard, inhibitors of protein– protein interactions have been successfully developed that target, amongst others, the interface of the large and small subunits of herpes simplex virus ribonucleotide reductase [8], cytokines (IL-2/IL-2Ra) [9], and growth hormone/receptor binding [10] The three enzymes of HIV (PR, RT and integrase (IN)) are all oligomeric proteins (Fig 1) The enzymatic functioning of each of these enzymes is entirely dependent on their quaternary structure [11–13] Therefore, inhibition of retroviral enzyme protein-protein assembly, or drug-mediated modulation of retroviral enzyme oligomers, provide alternative targets for HIV-1 inhibition
The objective of this review is to describe the unique structural features of the HIV-1 oligomeric enzymes PR, RT and IN and the strategies that have been developed to inhibit enzyme function by modulation of the interfaces between the subunits of the enzymes Each viral enzyme will
be dealt with individually
Correspondence to N Sluis-Cremer, Department of Medicine,
Division of Infectious Diseases, University of Pittsburgh, S808 Scaife
Hall, 3550 Terrace Street, Pittsburgh 15261, PA, USA.,
Fax: + 412 6489653, Tel.: + 412 3838525,
E-mail: CremerN@msx.dept-med.pitt.edu
Abbreviations: IN, integrase; NNRTI, non-nucleoside reverse
transcriptase inhibitor; PR, protease; RNase H, ribonuclease H; RT,
reverse transcriptase; TSAOe 3
T, 1-{spiro[4¢-amino-2¢,2¢-dioxo-1¢,2¢-oxathiole-5¢,3¢-[2¢,5¢-bis-O-(tert-butyldimethylsilyl)-b- D
-ribofurano-syl]]}-3-ethylthymine.
Enzymes: HIV-1 integrase (EC 2.7.7.49); HIV-1 protease
(EC 3.4.23.16); HIV-1 reverse transcriptase (EC 2.7.7.49); HIV-1
ribonuclease H(EC 3.1.26.4, SWISS-PROT entry name:
POL_HV1B1, Pol polyprotein of HIV-1 (BH10 isolate)).
(Received 22 May 2002, revised 28 June 2002,
accepted 29 August 2002)
Trang 2H I V - 1 P R O T E A S E
Structure and function of HIV-1 PR
HIV-1 PR catalyzes the hydrolysis of specific peptide bonds
within the HIV-1 Gag and Gag-Pol polyproteins to generate
the various structural and functional proteins essential for
viral replication HIV-1 PR is a symmetrically arranged
homodimeric protein composed of two chemically identical
subunits of 99 amino acids (Fig 1) The PR subunit fold
consists of a compact structure of b strands with a short
a helix near the C-terminus [14] The antiparallel b strands
constituted by residues 44–57 from both subunits, form a
flexible flap region that is thought to fold down over the
active site during catalysis to both bind substrate and
exclude water Protein–protein interactions in the dimer
include interactions between the catalytic triad residues
(D25-G27), I50 and G51 at the tip of the flaps, and the
antiparallel b sheet formed by the four termini in the dimer
(residues 1–5 and 95–99) Additional interactions include a
complex salt bridge between D29 and R87 of one subunit
and R8 of the other subunit Thermodynamic analyses of
the dimeric PR molecule indicates a Gibbs energy of dimer
stabilization of 10 kcal/mol at 25C (pH3.4), consistent
with a dissociation constant of 5· 10)8M [15]
Interest-ingly, the Gibbs free energy of dimerization is not uniformly
distributed along the protein–protein interface [15] Instead,
the interface is characterized by the presence of clusters of
residues (hot spots) that significantly contribute to subunit
association, and other regions that contribute very little In
particular, the four-stranded b sheet formed by the
amino-acid residues at the N- and C-termini of PR contribute close
to 75% of the total Gibbs energy [15] The importance of
this four-stranded b sheet is further emphasized by the fact
that all PR dimerization inhibitors developed by rational
(structure-assisted) design target this region (discussed below)
Peptide-based inhibitors of PR dimerization Short synthetic peptides corresponding to the amino-acid sequences of the N- and C-termini of HIV-1 PR have been shown to inhibit proteolytic activity by binding to the inactive PR subunits and preventing their association into active dimer [16–19] Peptides corresponding to the C-terminal segment of the HIV-1 matrix protein have also been found to elicit the same effect [20] However, the concentration of these peptides (both PR- and matrix-derived) required to effectively inhibit the PR monomer-dimer equilibrium by 50% (IC50) is relatively high (30–100 mM, Table 1) Schramm et al demonstrated that
it was possible to significantly improve their inhibitory properties ( 50–200 fold) through modification of their amino-acid composition and the addition of a hydrophobic moiety, such aminocaproyl or palmitoyl, to the N-terminus
of the peptide [20] The development of these more potent peptide inhibitors firmly established that PR dimerization was a rational target for the development of AIDS therapeutics, and that small-size peptide mimetics exhibiting good bio-availability could be derived for HIV-1 therapy In this regard, N-terminally palmitoyl-blocked peptides con-sisting of only three residues, one of which is a non-natural amino acid, have been developed and shown to exhibit good potency against PR dimerization [21]
Cross-linking of the N- and C-terminal peptides to form a mimic of the HIV)1 PR dimerization interface has provided an alternative strategy for the development of more potent PR dimerization inhibitors The principle of this strategy is illustrated in Fig 2 This approach was initially adopted by Babe et al., who cross-linked the N- and C-terminal PR-derived peptides by a 3.5-A˚ tether composed of three glycine residues, however, the resulting compounds were not potent inhibitors of PR dimerization [18] In the crystal structures of HIV-1 PR, the polypep-tide termini are held at a distance of approximately 10 A˚ (see Fig 2) Accordingly, more potent cross-linked inter-facial peptide compounds have been developed using tethers that bridge this gap [22–24] For example, Zutshi
et al used flexible alkyl-tethers to link the peptide strands [22], while Bouras et al took advantage of a
pyridinediol-or naphthalenediol-based scaffold [23] The supposed advantage of the aromatic conformationally constrained scaffold is that it may allow the two peptide strands to be initially more suitably oriented to permit formation of the antiparallel b sheet with one PR monomer [23] However, very little difference in relative potency is observed between the different tethers (Table 1) Irreversible inhibi-tion of PR dimerizainhibi-tion has also been achieved by designing a cross-linked interfacial peptide molecule that can form a disulfide bond with C95 in HIV-1 PR [25] Other novel strategies involve tethering an active-site peptide inhibitor with the dimerization inhibiting C-terminus peptide, thereby generating a compound that exhibits synergistic inhibition of PR activity [26]
The peptide inhibitors described above were all developed using peptide sequences corresponding to the N- and C-termini of PR, which themselves had been initially tested following the observation of their essential role in linking the
Fig 1 Oligomeric structures of HIV-1 PR (1A3O.pdb), RT
(1HMV.pdb)and IN (1EX4.pdb) The two subunits for each retroviral
enzyme are depicted in magenta and cyan, respectively Residues
contributing to the protein–protein interface are illustrated using a
surface representation.
Trang 3two PR subunits through the formation of the four-stranded
b sheet [14] Recently, an elegant strategy for the genetic
selection of dissociative peptide-based inhibitors of HIV-1
PR (and virtually all other designated protein–protein
interactions) has been reported [27] Briefly, this strategy
takes advantage of k-bacteriophage repressor protein (cI)
that binds to its operator (kPR) as a homodimer The
C-terminal dimerization domain of cI can be replaced by
another protein that homodimerizes, in this instance an
inactive variant of PR was used When bacteria are
transformed with a reporter plasmid (that contains the
selection module kPR-lacZ-tet and directs the production of
the cI-PR fusion protein), cI-PR represses the transcription
of the reporter genes and the transformants show a
LacZ(negative)-Tet(sensitive)phenotype [27]
Co-transfor-mation of the reporter plasmid with a peptide plasmid
library allows for the selection of peptides that prevent
NcI-PR dimerization and generate transformants exhibiting
a LacZ(positive)Tet(resistant) phenotype [27] The power
and utility of this technique was demonstrated by the
selection of approximately 300 peptides from 3· 108 cotransformants that exhibited a positive phenotype, rep-resenting a selection frequency of 1 in 106 Further analyses
of the selected peptides identified the peptide IVQVDAEGG
as an inhibitor of PR dimerization, which when tethered in a head-to-head or a tail-to-tail fashion generated a relatively potent inhibitor of PR dimerization (Table 1)
Non-peptide based inhibitors of PR dimerization
To date, two structurally unrelated classes of small-organic molecules have been identified which inhibit PR dimeriza-tion [28,29] The first class of molecules, which exhibit a polycyclic triterpene structure, were identified following a search of the Cambridge Structural Database (www.ccdc.cam.ac.uk) for pharmacophores that could bridge the 10 A˚ gap between the termini of a PR subunit [28] Extensive kinetic analysis of one of these triterpenes, ursolic acid, demonstrated that these compounds inhibited
PR dimerization with relatively high potency (K ¼ 3.4 lM)
Table 1 Peptide and small molecule inhibitors of HIV-1 PR dimerization.
IC 50 (lM) Method of analysis of PR dimerization Ref Peptides Derived from the
N-and C- Termini of HIV-1 PR N-and MA
Modified Peptides Derived from the
C-terminus of HIV-1 PR
Palmitoyl-Tyr-Glu-(p-biphenyl-alanine)-OH0.025 Kinetic analysis [21] Cross-linked Interfacial Peptides
2.0 Gel-filtration; protein cross-linking
PR fluorescence
[22]
Other Peptides
Kinetic analysis; gel-filtration Above peptide cross-linked using
Non-Peptide Based Inhibitors
a
Kinetic analyses were carried out according to the method described by Zhang et al (1991) [16].
Trang 4The second class of molecules that inhibited PR
dimeriza-tion include pentaester derivatives of of didemnaketal A
[29] The identification of these classes of small molecules is
significant, as in general many empirical searches for low
molecular mass pharmacological inhibitors (<400) of
protein–protein interactions have routinely failed
H I V - 1 R E V E R S E T R A N S C R I P T A S E
Structure and function of HIV-1 RT
HIV-1 RT is required for conversion of the viral genomic
RNA into a double-stranded proviral DNA precursor This
process is catalyzed by the RNA- and DNA-dependent
polymerase and ribonuclease H(RNase H) activities of the
enzyme in a reverse transcription complex in the cell
cytoplasm [30] HIV-1 RT is an asymmetric heterodimer
composed of a 560-residue 66 kDa subunit (p66)
compri-sing two domains termed DNA polymerase and RNase H,
and a p66-derived 440-residue 51 kDa subunit (p51) The
p51 subunit is produced during viral assembly and
matur-ation via HIV-1 protease-mediated cleavage of the
C-terminal (RNase H) domain of a p66 subunit [31] A
fascinating feature of the HIV-1 RT heterodimer is the
structural asymmetry which exists between the p66 and p51
subunits despite the fact that they are products of the same
gene and exhibit identical amino-acid sequences for the first
440 residues [32–40]
The overall shape of the p66 subunit has been likened to
that of a right-hand [35] The major subdomains of the
polymerase domain of p66 are termed fingers (residues
1–85, 118–155), palm (86–117, 156–237) and thumb (238–
318) The DNA polymerase catalytic aspartate residues
(D110, D185, and D186) reside in the palm subdomain A
fourth subdomain, termed the connection subdomain
(residues 319–426), acts as a tether between the DNA
polymerase and C-terminal RNase H(427–565) domains
The p51 subunit contains the same fingers, thumb, palm and connection subdomains, however, their spatial arrangement differs markedly to those of the p66 subunit [35]
Upon formation of the RT heterodimer from the p66 and p51 monomers, large surface areas of the individual subunits become inaccessible to water [33,41] Approxi-mately 4800 A˚2of protein surface is buried in the RT dimer complex of which 3050A˚2 corresponds to nonpolar atoms Amino-acid residues in the p66 subunit that form part of the dimer interface are derived primarily from the palm, connection and RNase Hdomain, while in the p51 they arise from the fingers, thumb and connection domains Dissection of the contributions of each individual residue to the total buried surface area upon dimerization reveals eight stretches of residues that make the largest contribution to total binding strength These include residues D86-L92, Q373-G384, W406-W410 and P537-G546 in p66 subunit, and P52-P55, I135-P140, C280-T290 and P392-W401 in the p51 subunit [41,42] Three clearly visible clusters are formed between these interfacial residues [33] A single region in the palm domain of p66 (D86-L92) interacts with two regions of the fingers of p51 (P52-P55 and S135-P140) The RNase H residues 537–546 in the p66 subunit interact with the p51 thumb residues 280–290, and the p66 connection residues W406-W410 interact with residues in the p51 connection domain residues (P392-W401) Evident from these clusters, the two subunits are completely asymmetric with respect to one another in that the subunit interface on p51 involves different amino acids than the p66 [33] Contacts between the connection subdomains form the only interactions between equivalent subdomains from each subunit How-ever, even in this case, many equivalent residues make different protein–protein interactions in such a way that the contacts between the two connection subdomains are also intrinsically asymmetric Thermodynamic evaluations of the association between the p66 and p51 subunits of RT have estimated a Gibbs free energy of dimer stabilization of approximately 10–12 kcalÆmol)1, corresponding to a disso-ciation constant of approximately 10 nM[43,44]
Peptide-based inhibitors of RT dimerization
As described above, one of the three clusters of residues formed between the RT p66 and p51 dimer interface are formed through the interactions between the RT p66 connection residues W406-W410 and residues P392-W401
in the p51 connection domain Interestingly, a 19 amino-acid synthetic peptide corresponding to residues 389–407 of the connection domain (N-FKLPIQKETWETWWTEYWQ-C)
of RT was demonstrated to be relatively efficient in retarding the heterodimerization process of HIV-1 RT [45] Further studies indicated that the same peptide was as efficient at inhibiting the heterodimerization process of HIV-2 RT as HIV-1 RT [46] This result is not surprising given that this region in the connection domain is conserved in 1,
HIV-2 and the simian immunodeficiency virus RTs [46] (the corresponding amino-acid sequence in HIV-2 RT is N-FHLPVERDTWEQWWDNYWQ-C) More recently, the length of the peptide was optimized to generate a shorter (10 residue) peptide (corresponding to residues 395–404 of RT) that was synthesized with an acetylated N-terminus and
a cysteamide group at the C-terminus to improve stability and cellular uptake [47] The resulting peptide was a more
Fig 2 Schematic representation of the strategy used to inhibit PR
dimerization by cross-linked interfacial peptides The N-terminus of PR
is indicated (N) The four-stranded b sheet formed by amino-acid
residues at the N- and C-termini of PR is a major binding determinant
in the formation of dimeric PR Cross-linked interfacial peptides
containing a tether region of approximately 10 A˚ inhibit PR
dimeri-zation by permitting the formation of a pseudo antiparallel b sheet
with one of the PR subunits.
Trang 5efficient inhibitor of RT dimerization in vitro and was also
shown to inhibit HIV-1 replication in cell culture [47] The
antiviral activity of the peptide was further enhanced by
conjugation to a peptidyl carrier without adverse toxic effects
to cells [47] Remarkably, the concentration of
peptide-carrier complex required to inhibit HIV-1 replication was
significantly less than the peptide concentration required for
the inhibition of RT dimerization in vitro For example,
0.1 nM of peptide-carrier completely suppressed HIV-1
replication for 15 days, whereas a peptide concentration of
240 mMwas required to inhibit RT heterodimerization by
50% in vitro [47] This may suggest that the mechanism of
inhibition of subunit association in vitro is different from the
process in HIV-1 infected cells In HIV-infected cells the RT
polypeptides are translated as part of the Gag–Pol
polypro-tein which is subsequently cleaved by HIV-1 PR to release
the various structural and functional proteins Recent studies
have shown that HIV-1 PR cleaves the Pol region of Gag–
Pol in a sequential manner in which the RT p66 polypeptide
is initially released from the polyprotein precursor Cleavage
of the p66 subunit to generate RT p51 appears to require a
p66/p66 homodimeric intermediate (D Arion, N
Sluis-Cremer & M.A Parniak, unpublished results) The
dissocia-tion constant for p66 homodimerizadissocia-tion is 10)6M, a value
approximately 1000-fold weaker than the interaction
be-tween RT p66 and p51 [43,44] Thus, one could anticipate
that the 10-residue peptide should be a more potent inhibitor
of p66 homodimerization Furthermore, it is interesting to
consider that modulation of RT dimerization may also affect
the interaction between two Gag–Pol molecules that must
dimerize to activate HIV-1 PR [48] Any affects on this
interaction may adversely affect PR activity [49] Hence, the
in vitro study of the effect of peptides on p66 and p51
dimerization does not necessarily reflect the process that is
occurring in HIV-infected cells and may not accurately
predict their impact on HIV-1 replication in cell based assays
Synthetic peptides that inhibit conformational changes
during HIV-1 RT heterodimerization
In vitroformation of active heterodimeric p66/p51 HIV-1
RT from the p66 and p51 monomeric subunits occurs in a
two step process involving an initial bimolecular association
followed by a slow conformational change [50] The
conformational change (or maturation step) appears to be
essential for the complete enzymatic activation of RT [50]
As discussed previously, the RNase Hresidues 537–546 in
the p66 subunit interact with the p51 thumb residues 280–
290 A synthetic peptide derived from a sequence within the
thumb subdomain of HIV-1 RT (residues 284–300) was
found to bind to heterodimeric HIV-1 RT with an apparent
dissociation constant in the nanomolar range and interfere
with the conformational change (or maturation step)
required for activation of heterodimeric RT [51] Based on
this work it was suggested that the activation of RT might
also represent an important target for the design of novel
antiviral compounds
Destabilization of the HIV-1 RT dimer interface
by small nonpeptidic molecules
The complete dissociation of the p66 and p51 subunits of
HIV-1 RT heterodimer may not be entirely necessary for
there to be a negative impact on RT enzymatic function Indeed, recent studies have shown that small molecule binding to the dimer interface of HIV-1 RT may induce conformational changes that impact on the overall stability
of the heterodimeric complex without dissociating the heterodimer complex [44,52] Two structurally unrelated classes of compounds have been found to elicit this effect 2¢,5¢-Bis-O-(tert-butyldimethylsilyl)-b-D -ribofuranosyl]-3¢spiro-5¢¢-(4¢¢-amino-1¢,2¢-oxathiole-2¢,2¢-dioxide)thymine (TSAO-T) is the prototype of an unusual class of non-nucleoside reverse transcriptase inhibitors (NNRTI) which have structures (Fig 3) and mechanism of actions quite distinct from conventional NNRTI [53,54] The N3-ethyl derivative of TSAO-T, TSAO-e3T has been shown to destabilize both the p66/p51 and p66/p66 dimeric forms of HIV-1 RT [44] The Gibbs free energy of RT dimer dissociation is decreased in the presence of increasing concentrations of TSAOe3T, resulting in loss of dimer stability of 4.0 and 3.2 kcalÆmol)1for p66/p51 and p66/p66 forms of HIV-1 RT, respectively [44] This loss of energy is not sufficient to induce subunit dissociation in the absence
of denaturant [44] High-level drug resistance to TSAO is mediated by the E138K mutation in the p51 subunit of HIV-1 RT [55] The introduction of this mutation into RT significantly diminishes the ability of TSAO to bind to and inhibit the enzyme [55] and accordingly TSAO-e3T is unable
to destabilize the subunit interactions of the E138K mutant enzyme [44] Modeling experiments have suggested that TSAO may bind to a site in RT that is overlapping with, but
Fig 3 Chemical structures of NNRTI that modulate the RT dimeri-zation process The NNRTI nevirapine, efavirenz, and UC781 act as chemical enhancers of HIV-1 RT dimerization [57] Unlike other NNRTI, delavirdine has no effect on RT dimerization [57] TSAOe3T and BBNHbinding to HIV-1 RT destabilizes the quaternary structure
of the enzyme [43,51].
Trang 6distinct from, the NNRTI binding site where it appears to
make significant interactions with the p51 subunit of the
enzyme [41,44] On the basis of this model, the
TSAO-induced changes in RT dimer stability likely arise from
conformational perturbations that affect the p66/p51 RT
interface [41,44]
N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde
hydrazone (BBNH) is a multitarget inhibitor of HIV-1 RT
that binds to both the DNA polymerase and RNase H
domains of the enzyme, and inhibits both enzymatic
activities [56,57] BBNHbinding to HIV-1 RT also impacts
on the dimeric stability of the heterodimeric enzyme [52] in
that BBNHbinding to p66/p51 RT decreases the value of
the Gibbs free energy of RT dimer dissociation by
3.8 kcalÆmol)1 To evaluate whether this loss of Gibbs free
energy was mediated by BBNHbinding to one or more sites
in RT, a variety of BBNHanalogs were synthesized and
evaluated for their ability to destabilize (or weaken) the
protein–protein interactions of the heterodimer [52] In this
regard, it was found that N-acyl hydrazone binding in the
DNA polymerase domain alone was sufficient to elicit the
observed decrease in Gibbs free energy In this regard, it has
been speculated that BBNHbinds to HIV-1 RT in a manner
analogous to TSAOe3T [52]
Small molecules that enhance RT dimerization
It is clear that either dissociation or destabilization of the
RT subunits is detrimental to enzyme function Conversely,
enhancement of the HIV)1 RT subunit interactions may
also represent a novel approach to modulating RT activity
In this regard, it has recently been reported that several
NNRTI exhibit an unexpected capacity to dramatically
increase the association of the p66 and p51 RT subunits [58]
Using a yeast two hybrid RT dimerization assay that
specifically detects the interaction between the p66 and p51
RT subunits [59] it was shown that several NNRTI,
including efavirenz, nevirapine, UC781, 8-Cl-TIBO, HBY
097 and a-APA, can significantly increase the
b-galactosi-dase readout in a yeast reporter strain [58] This increase in
b-galactosidase activity suggested an enhancement of RT
heterodimer subunit interaction, an effect that was
con-firmed by in vitro binding assays using recombinant p66 and
p51 [58] Enhanced homodimerization of the RT p66
subunits by efavirenz has also been observed in both the
Y2Hassay and in in vitro binding assays (G Tachedjian,
unpublished observations) Furthermore, this
NNRTI-induced enhancement effect on RT dimerization requires
drug binding to the NNRTI binding site in the p66 subunit
as introduction of the drug resistance mutation, Y181C, in
the NNRTI-binding pocket negates the enhancement effect
mediated by nevirapine [58] The mechanism by which these
small molecules enhance RT dimerization remains unclear
However, the mode of NNRTI binding to RT appears to be
important Delavirdine, also an NNRTI, does not enhance
RT dimerization [58] This drug, in contrast to other
NNRTI, is longer and does not sit exclusively in the
NNRTI binding pocket but protrudes outside this site [60]
The unique characteristics of the interaction of delaviridine
with the HIV-1 RT suggests that it binds to p66 in a way
that does not favor the enhancement of RT dimerization
[58] Elucidation of the differences in RT binding between
delavirdine and other NNRTI may provide important
information for the design of potent enhancers of RT dimerization and consequently potent inhibitors of DNA polymerization [58]
H I V - 1 I N T E G R A S E
Structure and function of HIV-1 IN HIV-1 IN is a polynucleotidyltransferase that catalyzes the integration of the DNA copy of the viral genome into the genome of the host cell In order to accomplish this goal, IN has evolved to catalyze two separate reactions, each proceeding by direct transesterification reactions catalyzed
at a single active site in the enzyme’s core [61] In the first reaction, 3¢ processing, IN removes two nucleotides from the from the 3¢-end of each strand of the nascent viral DNA, leaving a recessed 3¢CA dinucleotide After migra-tion into the nucleus of the infected cell as part of the nucleoprotein complex, IN covalently attaches each 3¢ processed viral end to the host cell DNA, a reaction termed strand transfer
HIV-1 IN comprises three independently folding domains; an N-terminal domain, a catalytic core domain, and a C-terminal domain (for a review see [62]) The N-terminal domain, residues 1–51, contains a conserved HH-CC motif that binds zinc in a 1 : 1 stoichiometry [63] The central catalytic core domain, residues 52–210, contains the catalytic site characterized by three invariant essential acidic residues, D64, D116 and E152 The C-terminal domain, residues 220–288, appears to significantly contrib-ute to DNA binding [64] and is linked to the catalytic core
by residues 195–220, an extension of the final helix of the core domain Efforts to crystallize the full length HIV-1 IN have been hampered by poor solubility However, the three-dimensional structure of each domain has been solved [65–67] as have structures of two domain INs containing either the catalytic core and C-terminal domain [68], or the N-terminal domain and the catalytic core [69] In all structures reported to date, the quaternary structure of IN is dimeric, however, the full enzyme is likely to function as at least a tetramer [13] The dimer interface in the catalytic core–C-terminal two domain fragment involves the strong helix-to-helix contacts a1 (residues 99–108):a5¢(residues 168–185) and a5:a1¢, where both hydrophobic and electro-static interactions contribute to dimer stabilization In the N-terminal –catalytic core two domain structure, additional subunit interface interactions are provided from the N-terminal domain, in particular residues 29–35
Peptide inhibitors of HIV-1 in oligomerization
As described above, protein–protein interactions between the two catalytic core domains involve interactions from the a1 and a5 helices of both subunits Synthetic peptides corresponding to the respective sequences (93–107 and 167– 187) were found to strongly inhibit the 3¢-processing and strand-transfer activities of IN [70] Furthermore, both peptides were found to perturb the association-dissociation equilibrium of both the full-length IN enzyme, as well as the individually isolated catalytic cores [70] Interestingly, peptide binding to IN also appeared to alter the overall conformation of the protein subunits, suggesting that enzyme deactivation, subunit dissociation and protein
Trang 7unfolding are events which parallel one another
Fluores-cence studies suggested that the peptide corresponding to
residues 167–187 physically interacts with helix a1 in the
dimer interface of the catalytic core domains thus providing
a rational for the observed dissociation of IN oligomers [70]
C O N C L U S I O N S
The enzymatic activities of HIV-1 PR, RT and IN are all
coupled to their quaternary (or oligomeric) structures
Accordingly, modulation of the protein–protein
inter-actions of these enzymes has been proposed as a rational
target for the development of anti-HIV compounds In this
regard, our review highlights the many peptidic and small
molecule compounds that have been identified to exhibit
such a mode of action However, in most cases, the
structural and kinetic characterization of their mechanisms
of action has primarily been carried out in an in vitro
environment, using recombinantly purified enzyme
Although some of the molecules described above have
been shown to exhibit antiviral activity in cell culture
[17,47,53,56], no studies have rigorously evaluated their
effect on either Gag-Pol processing or enzyme oligomer
formation in the virus Thus to date, there is essentially no
evidence to confirm that their mechanisms of action in vivo
are similar to those proposed in vitro In these authors’
opinions, such studies obviously represent the next logical
step in the unfolding story of the modulation of the
oligomeric structures of HIV-1 viral enzymes by synthetic
peptides and small molecules
A C K N O W L E D G E M E N T S
The authors would like to acknowledge Dominique Arion for critical
reading of the manuscript The research of N.S.-C has been funded, in
part, by a University of Pittsburgh Medical Center (UPMC)
Compet-itive Medical Research Fund (CMRF) G.T was supported in part by a
C.J Martin Fellowship 977373 awarded by the National Health and
Medical Research Council of Australia.
R E F E R E N C E S
1 Barre´-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T.,
Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C.,
Brun-Vezinet, F., Rouzioux, C., Rozembaum, W & Montagnier, L.
(1983) Isolation of a T-lymphotropic retrovirus from a patient at
risk for acquired immunodeficiency syndrome (AIDS) Science
220, 868–871.
2 Popovic, M., Sarngadharan, M.G., Read, E & Gallo, R.C (1984)
Detection, isolation and continuous production of cytoplasmic
retrovirus (HTLV-III) from patient with AIDS and pre-AIDS.
Science 224, 497–500.
3 Miller, M.D & Hazuda, D.J (2001) New antiretroviral agents:
looking beyond protease and reverse transcriptase Curr Opin.
Microbiol 4, 535–539.
4 Condra, J.H., Miller, M.D., Hazuda, D.J & Emini, E.A (2002)
Potential new therapies for the treatment of HIV-1 infection.
Annu Rev Med 53, 541–555.
5 Heldin, C.-H (1995) Dimerization of cell surface receptors in
signal transduction Cell 80, 213–223.
6 Gibson, W (1996) Structure and assembly of the virion
Inter-virology 39, 389–400.
7 Zutshi, R., Brickner, M & Chmielewski, J (1998) Inhibiting the
assembly of protein–protein interfaces Curr Opin Chem Biol 2,
62–66.
8 Liuzzi, M., Deziel, R., Moss, N., Beaulieu, P., Bonneau, A.-M., Bousquet, C., Chafouleas, J.G., Garneau, M., Jaramillo, J., Krogsrud, R.L et al (1994) A potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo Nature 372, 695–698.
9 Tilley, J.W., Chen, L., Fry, D.C., Emerson, S.D., Powers, G.D., Biondi, D., Varnell, T., Trilles, R., Guthrie, R., Mennoma, F., Kaplan, G., LeMahieu, R.A., Carson, M., Han, R.-J., Liu, C.-M., Palmermo, R & Ju, G (1997) Identification of a small molecule inhibitor of the IL-2/IL )2Ra receptor interaction which binds to IL-2 J Am Chem Soc 119, 7589–7590.
10 Judice, K (1997) Small molecule inhibitors of human growth hormone/receptor binding FASEB J 11, A839.
11 Babe, L.M., Pichuantes, S & Craik, C.S (1991) Inhibition of HIV protease activity by heterodimer formation Biochemistry 30, 106– 111.
12 Restle, T., Mu¨ller, B & Goody, R.S (1990) Dimerization of human immunodeficiency virus type 1 reverse transcriptase: a target for chemotherapeutic intervention J Biol Chem 265, 8986–8988.
13 Esposito, D & Craigie, R (1999) HIV integrase structure and function Adv Virus Res 52, 319–333.
14 Wlodawer, A., Miller, M., Jasko´lski, M., Sathyanarayana, B.K., Baldwin, E., Weber, I.T., Selk, L.M., Clawson, L., Schneider, J & Kent, S.B.H (1989) Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease Science 245, 616– 621.
15 Todd, M.J., Semo, N & Freire, E (1998) The structural stability
of the HIV-1 protease J Mol Biol 283, 475–488.
16 Zhang, Z.-Y., Poorman, R.A., Maggiora, L.L., Heinrikson, R.L.
& Ke´zdy, F.J (1991) Dissociative inhibition of dimeric enzymes.
J Biol Chem 266, 15591–15594.
17 Schramm, H.J., Nakashima, H., Schramm, W., Wakayama, H & Yamamoto, N (1991) HIV-1 reproduction is inhibited by peptides derived from the N- and C-termini of HIV-1 protease Biochem Biophys Res Commun 179, 847–851.
18 Babe´, L.M., Rose, J & Craik, C.S (1992) Synthetic interface peptides alter dimeric assembly of the HIV 1 and HIV 2 proteases Protein Sci 1, 1244–1253.
19 Franciskovich, J., Houseman, K., Mueller, R & Chmielewski, J (1993) A systematic evaluation of the inhibition of HIV-1 protease
by its C- and N-terminal peptides Bioorg Med Chem Lett 3, 765–768.
20 Schramm, H.J., Boetzel, J., Bu¨ttner, J., Fritsche, E., Go¨hring, W., Jaeger, E., Ko¨nig, S., Thumfart, O., Wenger, T., Nagel, N.E & Schramm, W (1996) The inhibition of human immunodeficiency virus proteases by interface peptides Antiviral Res 30, 155–170.
21 Schramm, H.J., de Rosny, E., Reboud-Ravaux, M., Bu¨ttner, J., Dick, A & Schramm, W (1999) Lipopeptides as dimerization inhibitors of HIV-1 protease Biol Chem 380, 593–596.
22 Zutshi, R., Franciskovich, J., Shultz, M., Schweitzer, B., Bishop, P., Wilson, M & Chmielewski, J (1997) Targeting the dimerization interface of HIV-1 protease: Inhibition with the cross-linked interfacial peptides J Am Chem Soc 119, 4841– 4845.
23 Bouras, A., Boggetto, N., Benatalah, Z., de Rosny, E., Sicsic, S & Reboud-Ravaux, M (1999) Design, synthesis, and evaluation of conformationally constrained tongs, new inhibitors of HIV-1 protease dimerization J Med Chem 42, 957–962.
24 Song, M., Rajesh, S., Hayashi, Y & Kiso, Y (2001) Design and synthesis of new inhibitors of HIV-1 protease dimerization with conformationally constrained templates Bioorg Med Chem Lett.
11, 2465–2468.
25 Zutshi, R & Chmielewski, J (2000) Targeting the dimerization interface for irreversible inhibition of HIV-1 protease Bioorg Med Chem Lett 10, 1901–1903.
Trang 826 Uhlı´kova´, T., Konvalinka, J., Pichova´, Soucek, M., Kra¨usslich,
H.-G & Vondra´sek, J (1996) A modular approach to HIV-1
proteinase inhibitor design Biochem Biophys Res Commun 222,
38–43.
27 Park, S.-H & Raines, R.T (2000) Genetic selection for
dis-sociative inhibitors of designated protein–protein interactions.
Nat Biotechnol 18, 847–851.
28 Que´re´, L., Wenger, T & Schramm, H.J (1996) Triterpenes as
potential dimerization inhibitors of HIV-1 protease Biochem.
Biophys Res Commun 227, 484–488.
29 Fan, X., Flentke, G.R & Rich, D.H (1998) Inhibition of H IV-1
protease by a subunit of didemnaketal A J Am Chem Soc 120,
8893–8894.
30 Telesnitsky, A & Goff, S (1997) Reverse transcriptase and the
generation of retroviral DNA Retroviruses (Coffin, J., Hughes, S.
& Varmus, H., eds), pp 121–160 Cold Spring Harbor Laboratory
Press, Plainview, NY.
31 di Marzo Veronese, F., Copeland, T.D., DeVico, A.L., Rahman,
R., Oroszlan, S., Gallo, R.C & Sarngadharan, M.G (1986)
Characterization of highly immunogenic p66/p51 as the reverse
transcriptase of HTLV-III/LAV Science 231, 1289–1291.
32 Hsiou, Y., Ding, J., Das, K., Clark, A.D Jr, Hughes, S.H &
Arnold, E (1996) Structure of unliganded HIV-1 reverse
tran-scriptase at 2.7 A˚ resolution: implications of conformational
changes for polymerization and inhibition mechanisms Structure
4, 853–860.
33 Wang, J., Smerdon, S.J., Jager, J., Kohlstaedt, L.A., Rice, P.A.,
Friedman, J.M & Steitz, T.A (1994) Structural basis of
asym-metry in the human immunodeficiency virus type 1 reverse
tran-scriptase heterodimer Proc Natl Acad Sci USA 91, 7242–7246.
34 Rodgers, D.W., Gamblin, S.J., Harris, B.A., Ray, S., Culp, J.S.,
H ellmig, B., Woolf, D.J., Debouck, C & H arrison, S.C (1995)
The structure of unliganded reverse transcriptase from the human
immunodeficiency virus type 1 Proc Natl Acad Sci USA 92,
1222–1226.
35 Kohlsteaedt, L.A., Wang, J., Friedman, J.M., Rice, P.A & Steitz,
T.A (1992) Crystal structure of 3.5 A˚ resolution of HIV-1 reverse
transcriptase complexed with an inhibitor Science 256, 1783–
1790.
36 Ren, J., Esnouf, R., Garman, E., Somers, D., Ross, C., Kirby, I.,
Keeling, J., Darby, G., Jones, Y., Stuart, D & Stammers, D.
(1995) High resolution structures of HIV-1 RT from four
RT-inhibitor complexes Nat Struct Biol 2, 293–302.
37 Das, K., Ding, J., H siou, Y., Clark, A.D Jr, Moereels, H ,
Koy-mans, L., Andries, K., Pauwels, R., Janssen, P.A., Boyer, P.L.,
Clark, P., Smith, R.H Jr, Kroeger Smith, M.B., Michejda, C.J.,
H ughes, S.H & Arnold, E (1996) Crystal structures of 8-Cl and
9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO
complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant.
J Mol Biol 264, 1085–1100.
38 Jacobo-Molina, A., Ding, J., Nanni, R.G., Clark, A.D Jr, Lu, X.,
Tantillo, C., Williams, R.L., Kamer, G., Ferris, A.L., Clark, P.,
Hizi, A., Huges, S.H & Arnold, E (1993) Crystal structure of
human immunodeficiency virus type 1 reverse transcriptase
com-plexed with double-stranded DNA at 3.0 A˚ resolution shows bent
DNA Proc Natl Acad Sci USA 90, 6320–6324.
39 Sarafianos, S.G., Das, K., Tantillo, C., Clark, A.D Jr, Ding, J.,
Whitcomb, J.M., Boyer, P.L., Hughes, S.H & Arnold, E (2001)
Crystal structure of HIV-1 reverse transcriptase in complex with a
polypurine tract RNA: DNA EMBO J 20, 1449–1461.
40 Huang, H., Chopra, R., Verdine, G.L & Harrison, S.C (1998)
Structure of a covalently trapped catalytic complex of HIV-1
reverse transcriptase: implications for drug resistance Science 282,
1669–1675.
41 Rodrı´guez-Barrios, F., Pe´rez, C., Lobato´n, E., Vela´zquez, S.,
Chamorro, C., San-Fe´lix, A., Pe´rez-Pe´rez, M.J., Camarasa,
M.J., Pelemans, H., Balzarini, J & Gago, F (2001) Identifica-tion of a putative binding site for [2¢,5¢-Bis-O-(tert-butyldi-methylsilyl)-b- D -ribofuranosyl]-3¢-spiro-5¢-(4¢-amino-1¢,2¢-oxathi-ole-2¢,2¢-dioxide) thymine (TSAO) derivatives at the p51–p66 interface of the HIV-1 reverse transcriptase J Med Chem 44, 1853–1865.
42 Mene´ndez-Arias, L., Abraha, A., Quinones-Mateu, M.E., Mas, A., Camarasa, M.J & Arts, E.J (2001) Functional characteriza-tion of chimeric reverse transcriptases with polypeptide subunits of highly divergent HIV-1 group M and O strains J Biol Chem 276, 27470–27479.
43 Divita, G., Rittinger, K., Restle, T., Immendorfer, U & Goody, R.S (1995) Conformational stability of dimeric HIV-1 and HIV-2 reverse transcriptases Biochemistry 34, 16337–16346.
44 Sluis-Cremer, N., Dmitrienko, G.I., Balzarini, J., Camarasa, M.J.
& Parniak, M.A (2000) Human immunodeficiency virus type 1 reverse transcriptase dimer destabilization by 1-[spiro[4¢-amino- 2¢,2¢-dioxo-1¢,2¢-oxathiole-5¢,3¢-[2¢,5¢-bis-O-(tert-butyldimethylsi-lyl)-beta- D -ribofuranosyl]]]-3-ethylthymine Biochemistry 39, 1427–1433.
45 Divita, G., Restle, T., Goody, R.S., Chermann, J.C & Baillon, J.G (1994) Inhibition of human immunodeficiency virus type
1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain J Biol Chem 269, 13080– 13083.
46 Divita, G., Baillon, J.G., Rittinger, K., Chermann, J.C & Goody, R.S (1995) Interface peptides as structure-based human immunodeficiency virus reverse transcriptase inhibitors J Biol Chem 270, 28642–28646.
47 Morris, M.C., Robert-H ebmann, V., Chaloin, L., Mery, J., H eitz, F., Devaux, C., Goody, R.S & Divita, G (1999) A new potent HIV-1 reverse transcriptase inhibitor A synthetic peptide derived from the interface subunit domains J Biol Chem 274, 24941– 24946.
48 Swanstrom, R & Wills, J (1997) Synthesis, assembly, and pro-cessing of viral proteins In Retroviruses (Coffin, J., Hughes, S & Varmus, H., eds), pp 263–334 Cold Spring Harbor Laboratory Press, Plainview, NY.
49 YuQ., Ottmann, M., Pechoux, C., Le Grice, S & Darlix, J.L (1998) Mutations in the primer grip of human immunodeficiency virus type 1 reverse transcriptase impair proviral DNA synthesis and virion maturation J Virol 72, 7676–7680.
50 Divita, G., Rittinger, K., Geourjon, C., Deleage, G & Goody, R.S (1995) Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process J Mol Biol 245, 508–521.
51 Morris, M.C., Berducou, C., Mery, J., H eitz, F & Divita, G (1999) The thumb domain of the p51-subunit is essential for ac-tivation of HIV reverse transcriptase Biochemistry 38, 15097– 15103.
52 Sluis-Cremer, N., Arion, D & Parniak, M.A (2002) Destabili-zation of the HIV-1 reverse transcriptase dimer upon interaction with N-acyl hydrazone inhibitors Mol Pharmacol 62, 398–405.
53 Balzarini, J., Pe´rez-Pe´rez, M.J., San-Fe´lix, A., Schols, D., Perno, C.F., Vandamme, A.M., Camarasa, M.J & De Clercq, E (1992) 2¢,5¢-Bis-O-(tert-butyldimethylsilyl)-3¢-spiro-5¢¢-(4¢¢-amino-1¢¢,2¢¢-oxathiole-2¢¢,2¢-dioxide) pyrimidine (TSAO) nucleoside analogues: highly selective inhibitors of human immunodeficiency virus type 1 that are targeted at the viral reverse transcriptase Proc Natl Acad Sci USA 89, 4392–4396.
54 Balzarini, J., Pe´rez-Pe´rez, M.J., San-Fe´lix, A., Camarasa, M.J., Bathurst, I.C., Barr, P.J & De Clercq, E (1992) Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue [2¢,5¢-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofurano-syl]-3¢-spiro-5¢-(4¢-amino-1¢,2¢-oxathiole-2¢,2¢-dioxide) thymine (TSAO-T) J Biol Chem 267, 11831–11838.
Trang 955 Jonckheere, H., Taymans, J.M., Balzarini, J., Velazquez, S.,
Camarasa, M.J., Desmyter, J., De Clercq, E & Anne, J (1994)
Resistance of HIV-1 reverse transcriptase against
[2¢,5¢-bis-O-(tert-
butyldimethylsilyl)-3¢-spiro-5¢¢-(4¢¢-amino-1¢¢,2¢¢-oxathiole-2¢¢,2¢¢-dioxide)] (TSAO) derivatives is determined by the mutation
Glu138 fi Lys on the p51 subunit J Biol Chem 269, 25255–
25258.
56 Borkow, G., Fletcher, R.S., Barnard, J., Arion, D., Motakis, D.,
Dmitrienko, G.I & Parniak, M.A (1997) Inhibition of the
ribo-nuclease Hand DNA polymerase activities of H IV-1 reverse
transcriptase by
N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthalde-hyde hydrazone Biochemistry 36, 3179–3185.
57 Arion, D., Sluis-Cremer, N., Min, K.-L., Abram, M.E., Fletcher,
R.S & Parniak, M.A (2002) Mutational analysis of Tyr-501 of
HIV-1 reverse transcriptase Effects on ribonuclease H activity and
inhibition of this activity by N-acylhydrazones J Biol Chem 277,
1370–1374.
58 Tachedjian, G., Orlova, M., Sarafianos, S.G., Arnold, E & Goff,
S.P (2001) Nonnucleoside reverse transcriptase inhibitors are
chemical enhancers of dimerization of the HIV type 1 reverse
transcriptase Proc Natl Acad Sci USA 98, 7188–7193.
59 Tachedjian, G., Aronson, H.E & Goff, S.P (2000) Analysis of
mutations and suppressors affecting interactions between the
subunits of the HIV type 1 reverse transcriptase Proc Natl Acad.
Sci USA 97, 6334–6339.
60 Esnouf, R.M., Ren, J., Hopkins, A.L., Ross, C.K., Jones, E.Y.,
Stammers, D.K & Stuart, D.I (1997) Unique features in the
structure of the complex between HIV-1 reverse transcriptase and
the bis (heteroaryl) piperazine (BHAP) U-90152 explain resistance
mutations for this nonnucleoside inhibitor Proc Natl Acad Sci.
USA 94, 3984–3989.
61 Engelman, A., Mizuuchi, K & Craigie, R (1991) HIV-1 DNA
integration: Mechanism of viral DNA cleavage and DNA strand
transfer Cell 67, 1211–1221.
62 Asante-Appiah, E & Skalka, A.M (1999) HIV-1 integrase: Structural organization, conformational changes, and catalysis Adv Virus Res 52, 351–369.
63 Zheng, R., Jenkins, T.M & Craigie, R (1996) Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization and enhances activity Proc Natl Acad Sci USA 93, 13659– 13664.
64 Brown, P (1997) Integration In Retroviruses (eds Coffin, S., Hughes, S.H & Varmus, H.E.), pp 161–203 Cold Spring Harbor Laboratory Press, Plainview, NY.
65 Cai, M., Zheng, R., Caffrey, R., Clore, M & Gronenborn, A.M (1997) Solution structure of N-terminal zinc binding domain of HIV-1 integrase Nat Struct Biol 4, 567–577.
66 Dyda, F., Hickman, A.B., Jenkins, T.M., Engelman, A., Craigie,
R & Davies, D.R (1994) Crystal structure of the catalytic domain
of HIV-1 integrase: Similarity to other polynucleotidyl trans-ferases Science 266, 1981–1986.
67 Lodi, P.J., Ernst, J.A., Kuszewski, J., H ickman, A.B., Engelman, A., Craigie, R., Clore, G.M & Gronenborn, A.M (1995) Solution structure of the DNA binding domain of HIV-1 integrase Biochemistry 34, 9826–9833.
68 Chen, J.C.-H., Krucinski, J., Miercke, L.J.W., Finer-Moore, J.S., Tang, A.H , Leavitt, A.D & Stroud, R.M (2000) Crystal struc-ture of the HIV-1 integrase catalytic core and C-terminal domains:
a model for viral DNA binding Proc Natl Acad Sci USA 97, 8233–8238.
69 Wang, J.-Y., Ling, H., Yang, W & Craigie, R (2001) Structure of
a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein EMBO J 20, 7333– 7343.
70 Maroun, R.G., Gayet, S., Benleulmi, M.S., Porumb, H., Zargar-ian, L., Merad, H , Leh, H , Moucadet, J.-L., Troalen, F & Fermandjian, S (2001) Peptide Inhibitors of HIV-1 integrase dissociate the enzyme oligomers Biochemistry 40, 13840–13848.