Báo cáo Y học: Modulation of the oligomeric structures of HIV-1 retroviral enzymes by synthetic peptides and small molecules pptx

REVIEW ARTICLE
Modulation of the oligomeric structures of HIV-1 retroviral enzymes
by synthetic peptides and small molecules
Nicolas Sluis-Cremer
1
and Gilda Tachedjian
2
1
Department of Medicine, Division of Infectious Diseases, University of Pittsburgh, PA, USA;
2
AIDS 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 additional 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:
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)
Eur. J. Biochem. 269, 5103–5111 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03216.x
HIV-1 PROTEASE
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 ÔflapÕ 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 25 °C (pH 3.4), consistent
with a dissociation constant of 5 · 10
)8
M
[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 (Ôhot spotsÕ) 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 ÔrationalÕ
(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% (IC
50
) is relatively high
(30–100 m
M
,Table1).Schrammet 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 Ômore potentÕ
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 Ôconformationally constrainedÕ
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 dimerization 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.
5104 N. Sluis-Cremer and G. Tachedjian (Eur. J. Biochem. 269) Ó FEBS 2002
two 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 (kP
R
) 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 kP
R
-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 · 10
8
cotransformants that exhibited a Ôpositive phenotypeÕ,rep-

resenting a selection frequency of 1 in 10
6
. 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
i
¼ 3.4 l
M
).
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 and MA

Ac-Thr-Leu-Asn-Phe-COOH 45 Kinetic analysis
a
[16]
N-Pro-Gln-Ile-Thr-Leu-Trp-OH >100 Kinetic analysis [19]
Ac-Gln-Val-Ser-Gln-Asn-Tyr-COOH 100 Kinetic analysis [20]
Modified Peptides Derived from the
C-terminus of HIV-1 PR
Thr-Val-Ser-Tyr-Glu-Leu-OH 12 Kinetic analysis [20]
Palmitoyl-Thr-Val-Ser-Tyr-Glu-Leu-OH 0.5 Kinetic analysis [20]
Palmitoyl-Tyr-Glu-Leu-OH 0.15 Kinetic analysis [21]
Palmitoyl-Tyr-Glu-(
L
-threonine)-OH 0.05 Kinetic analysis [21]
Palmitoyl-Tyr-Glu-(p-biphenyl-alanine)-OH 0.025 Kinetic analysis [21]
Cross-linked Interfacial Peptides
>50 Protein cross-linking [18]
2.0 Gel-filtration; protein cross-linking
PR fluorescence
[22]
4.2 Kinetic analysis [23]
Other Peptides
Ile-Val-Gln-Val-Asp-Ala-Glu-Gly-Gly 32 Genetic selection [27]
Kinetic analysis; gel-filtration
Above peptide cross-linked using
1,6-hexane-bis-vinylsulfone 0.78 Kinetic analysis [27]
Non-Peptide Based Inhibitors
Ursolic acid 3.4 Kinetic analysis [28]
Didemnaketal A penta-ester derivative 2.1 Kinetic analysis [29]
a
Kinetic analyses were carried out according to the method described by Zhang et al. (1991) [16].

Ó FEBS 2002 Modulation of the quaternary structure of HIV-1 enzymes (Eur. J. Biochem. 269) 5105
The 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.
HIV-1 REVERSE TRANSCRIPTASE
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 Ôright-handÕ [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 ÔconnectionÕ 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
˚
2
of 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 H domain, 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 n
M
[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 HIV-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.
5106 N. Sluis-Cremer and G. Tachedjian (Eur. J. Biochem. 269) Ó FEBS 2002
efficient 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 n
M
of peptide-carrier completely suppressed HIV-1
replication for 15 days, whereas a peptide concentration of
240 m
M
was 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 homodimerization is 10
)6
M
, 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 vitro formation 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 H residues 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-e
3
T 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 TSAOe
3
T, resulting in loss of dimer
stability of 4.0 and 3.2 kcalÆmol
)1
for 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-e
3
T 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]. TSAOe
3
T
and BBNH binding to HIV-1 RT destabilizes the quaternary structure
of the enzyme [43,51].
Ó FEBS 2002 Modulation of the quaternary structure of HIV-1 enzymes (Eur. J. Biochem. 269) 5107
distinct 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]. BBNH binding to HIV-1 RT also impacts
on the dimeric stability of the heterodimeric enzyme [52] in
that BBNH binding 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 BBNH binding to one or more sites
in RT, a variety of BBNH analogs 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 BBNH binds to HIV-1 RT in a manner
analogous to TSAOe
3
T[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
Y2H assay 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].
HIV-1 INTEGRASE

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
a1anda5 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
5108 N. Sluis-Cremer and G. Tachedjian (Eur. J. Biochem. 269) Ó FEBS 2002
unfolding 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].

CONCLUSIONS
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.
ACKNOWLEDGEMENTS
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.

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