Tài liệu Báo cáo khoa học: The capsid protein of human immunodeficiency virus: designing inhibitors of capsid assembly ppt

MINIREVIEW
The capsid protein of human immunodeficiency
virus: designing inhibitors of capsid assembly
Jose
´
L. Neira
1,2
1 Instituto de Biologı
´
a Molecular y Celular, Universidad Miguel Herna
´
ndez, Alicante, Spain
2 Biocomputation and Complex Systems Physics Institute, Zaragoza, Spain
Introduction
HIV-1, the agent responsible for AIDS, belongs to the
retrovirus family. Its genome is formed by two copies
of a single-stranded RNA, which are encased within a
lipoprotein shell, together with several replicative and
accessory proteins. The virus genome is a model of
economic packaging because the virus uses complex
processing (both in the production and cleavage of
mRNAs and in the final viral polypeptides) to
generate several proteins that can work in the host
cell. Perhaps the most important example of this eco-
nomic architecture is the production of Gag and Gag-
Pol fusion proteins.
The GagPol fusion protein, formed by a ribosomal
frame shift event, is cleaved into the Gag and Pol
polypeptides by the viral protease. Further cleavage
of the Pol chain yields the viral integrase (p31), a
new protease (p10), the reverse transcriptase (p50)

and RNase H (p15). Conversely, cleavage of the
Keywords
CTD protein; HIV; NMR; organic molecules;
protein–peptide interactions; protein–protein
interactions; structure; X-ray crystallography
Correspondence
J. L. Neira, Instituto de Biologı
´
a Molecular y
Celular, Edificio Torregaita
´
n, Universidad
Miguel Herna
´
ndez, Avda. del Ferrocarril s ⁄ n,
03202, Elche (Alicante), Spain
Fax: +34 9666 58758
Tel: +34 9666 58459
E-mail:
Note
The author would like to dedicate this paper
to Professors Manuel Rico and Alan Fersht
for allowing him to learn beside them
(Received 9 February 2009, revised 2 July
2009, accepted 24 July 2009)
doi:10.1111/j.1742-4658.2009.07314.x
The mature capsid of human immunodeficiency virus, HIV-1, is formed by
the assembly of copies of a capsid protein (CA). The C-terminal domain of
CA, CTD, is able to homodimerize and most of the dimerization interface
is formed by a single a-helix from each monomer. Assembly of the HIV-1

capsid critically depends on CA–CA interactions, including CTD interac-
tion with itself and with the CA N-terminal domain, NTD. This minireview
reports on the search and the design of peptides and small organic com-
pounds that are able to interact with the CTD and ⁄ or CA of HIV-1. Such
molecules aim to disrupt and ⁄ or alter the oligomerization capability of
CTD. The different peptides designed so far interact with CTD mainly via
hydrophobic contacts with residues close or belonging to the interface
between the dimerization helices. A CTD-binding organic compound also
establishes hydrophobic contacts with regions involved in the interface
between the NTD and CTD. These results open new venues for the devel-
opment of new antiviral drugs that are able to interact with CA and ⁄ or its
domains, hampering HIV-1 assembly and infection.
Abbreviations
CA, capsid protein of HIV-1 (p24); CAC1, a CTD-binding designed peptide; CAI, a CTD-binding phage display peptide; CAP-1, N-(3-chloro-4-
methylphenyl)-N¢-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl] ethyl}-urea); CTD, C-terminal domain of CA, comprising residues
146–231 of the intact protein; CTDW184A ⁄ M185A, a double mutant of CTD with Ala substitutions at the positions Trp184 and Met185;
HSQC, heteronuclear single quantum coherence; MA, matrix protein of HIV-1; NC, nucleocapsid protein; NTD, N-terminal domain of CA,
comprising residues 1–145 of the intact protein; NYAD-1, an improved designed version of CAI.
6110 FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS
Gag polyprotein, yields the matrix protein (MA),
the capsid protein (CA), the nucleocapsid protein
(NC) and p6 proteins, as well as two spacer pep-
tides.
Currently, most of the available drugs in the market
against AIDS target the reverse transcriptase and pro-
tease enzymes, produced by Pol. In general, the com-
pounds involved in the treatment of HIV infection
belong to one of the following types: (a) non-nucleo-
tide reverse transcriptase inhibitors; (b) nucleotide
reverse transcriptase inhibitors; and (c) protease inhibi-

tors [1]. Because of the poorly understood side-effects
of protease inhibitors in human fat metabolism [2],
and the frequent emergence and spread of drug-resis-
tant variants of HIV-1 [3], it is necessary to identify
new types of drugs that are suitable for long-term
usage, which can, at the very least, supplement the cur-
rently available drug regimes. Some of those new drug
candidates have been reviewed elsewhere [1,4] and their
pros and cons for possible anti-HIV chemotheraphy
have been discussed; this minireview focuses on CA as
a possible target for new anti-HIV-1 drugs. Among its
other biophysical features [5], the suitability of CA as
a drug target is based on: (a) its own self-assembly
properties [6]; and (b) the other protein–protein inter-
actions where it is involved [7]. CA in solution is a
homodimer with a dissociation constant of 10–18 lm [8].
Homo-oligomerization provides several structural and
functional advantages to proteins, such as an improved
stability or modular complex formation, whereas the
cell minimizes the genome size; indeed, oligomerization
is generally employed in nature to build viral capsids,
with a minimum of genetic information [9–13]. Fur-
thermore, because of the omnipresent nature of pro-
tein–protein interactions in key steps during the virus
life cycle, it is possible to design antiviral strategies
based on the inhibition of those active macromolecular
complexes [14]. This minireview focuses on the devel-
opment of small molecules (peptides and organic mole-
cules) that are potentially able to inhibit HIV-1
infection by impairing the self-assembly of CA. Several

peptides and one organic compound have been found
to inhibit the C-terminal domain of CA (CTD) dimer-
ization or, alternatively, its interactions with the N-ter-
minal domain of CA (NTD). This minireview focuses
on the structure of the complexes formed by CTD and
those molecules, as well as what can be learnt from
such studies. The use of drugs such as Bevirimat
(Panacos Pharmaceuticals, Watertown, MA, USA)
(although it is in Phase II trials) is not discussed
because its mechanism of action is not based on inhibi-
tion of CA self-assembly. Bevirimat is a novel HIV-1
maturation inhibitor that inhibits specifically the final
rate-limiting step in Gag processing; the drug prevents
the release of mature capsid protein from its precursor,
resulting in the production of immature, non-infectious
virus particles [15].
Before describing how those designed molecules
work and inhibit dimer formation, it is necessary to
describe in some detail the domain architecture of CA
of HIV-1. The CA protein is formed by two indepen-
dently folded domains, NTD and CTD, separated by a
flexible linker [8,16–18]; see also Fig. 1B in Mascare-
nhas and Musier-Forsyth [7]. The NTD (residues
1–146 in the numbering of the whole intact protein) is
composed of five coiled-coil a-helices (helices 1–5 of
CA), with two additional short a-helices (helices 6 and
7) following an extended proline-rich loop [16–18]. The
CTD domain (residues 147–231) is a dimer both in
solution and in the crystal form [8,19]. Each CTD
monomer is composed of a short 3

10
-helix followed by
a strand and four a-helices (helices 8–11 of CA):
a-helix 8 (residues 160–172), a-helix 9 (residues 178–
191), a-helix 10 (residues 195–202) and a-helix 11 (resi-
dues 209–214), which are connected by short loops or
turn-like structures (Fig. 1). The dimerization interface
of CTD is formed by the mutual docking of a-helix 9
from each monomer, with the side chains of each tryp-
tophan (Trp184) deeply buried in the dimer interface
[8,19]; this helix has a kink at Thr188. The two addi-
tional aromatic residues in each monomer, Tyr164 and
Fig. 1. Structure of the CTD dimer. X-ray structure of CTD showing
the dimeric structure of the domain. The monomers are depicted in
the same colour (blue) and the dimerization helix is highlighted
(gold), with the side-chain of the sole trypotophan in each monomer
indicated by sticks. The figure was produced using
PYMOL (http://
www.pymol.org) [39] using the Protein Data Bank file for CTD
(accession no. 1a43) [8]. The different a-helices and the kink at the
second one are indicated; the numbering of these helices corre-
sponds to helices 8–11 of the whole intact CA.
J. L. Neira CA small-molecule interactions
FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS 6111
Tyr169, are located in the hydrophobic core of each
monomer, well away from the dimer interface. The
dissociation constant of CTD is 10–18 l
M [8], similar
to that of intact CA (see above). Thus, the CA dimer-
ization interface is fully contained within the isolated

CTD. This observation has facilitated the initial search
for inhibitors of HIV-1 assembly by using simplified
experimental setups in vitro; if a small molecule is able
to dissociate CTD dimers in solution, then it could be
considered a potential inhibitor of CA assembly and
HIV-1 infectivity (dependent on that hypothesis always
being tested by direct experiments). Furthermore, the
same observation validates the view that the structures of
CTD small-molecule complexes can be physiologically
relevant, and thus they could provide important guide-
lines for the rational design of better assembly inhibitors,
as well as physiologically acceptable antiviral drugs.
Inhibition of CTD self-assembly by
peptides
Protein assembly processes can be considered as good
targets for antivirals because they depend only on recur-
ring weak interprotein interactions, and the disruption
of only a few of these interactions may be sufficient to
suppress infectivity. In principle, destabilization of the
CA dimeric species and, subsequently, HIV capsid
assembly can be carried out by using isolated CTD as a
competitive inhibitor [20,21]. Alternatively, virion
assembly can be hampered by the use of small peptides
or organic molecules which bind to: (a) CTD (or CA)
monomers and occupy the dimerization interface or
(b) nearby sites to the dimerization interface, thus weak-
ening the binding to the other monomer. Below, the
structural implications of peptides that are able to bind
to CTD are reviewed, as well as how they work based
on the reported structural studies.

There was an early attempt to design peptides that
could hamper dimerization of the whole CA in solu-
tion [22]. The peptide with the highest affinity for the
whole CA has the sequence IPVGEIYKRW, which
corresponds to the a-helix 7 of NTD. This region is
important for the formation of CA hexamers [6]; there-
fore, this peptide could inhibit the formation of the
hexamers, and assembly of the HIV-1 capsid, but no
evidence is yet available.
A peptide-mimicking the dimerization-helix of
CTD as an inhibitor of CA dimerization
Both structural [8] and thermodynamic [23] analyses
indicate that most of the dimerization interface in CA
(Fig. 1) is contained within a single helical segment
(helix 9). Thus, it was reasonable to assume, as a first
approach, that a synthetic peptide mimicking the
sequence of helix 9 could constitute a good inhibitor
of CTD dimerization. Accordingly, our group designed
a peptide comprising the dimerization helix of CTD,
plus three residues on each site to avoid fraying effects:
the sequence of this peptide, CAC1, is: Ac-EQ-
ASQEVKNWMTETLLVQNA-CONH
2
[24]. We first
tested whether CAC1 was bound to CTD in solution
by using several biophysical techniques. Thermal dena-
turations followed by CD, NMR and size-exclusion
chromatography provided evidence for the interaction
between CAC1 and CTD. Gel filtration analysis also
provided evidence for dissociation of the CTD dimer

mediated by the peptide because the protein eluted at
larger volumes in the presence of equimolar peptide
concentrations.
A quantitative value of the affinity constant of the
CAC1–CTD complex was determined by fluorescence
(using an anthraniloyl-labeled peptide), affinity chro-
matography and isothermal titration calorimetry. The
three techniques yielded similar values for the apparent
dissociation constant of the complex, in the order of
50 lm, which is only three- to five-fold higher than the
dissociation constant of dimeric CTD (10–18 lm) [8].
Because CAC1 is random-coil when isolated in solu-
tion, as shown by 1D-NMR experiments, it is impor-
tant to note that the above determined affinity
constant must take into account the entropy penalty to
fold the peptide. In view of this fact, and the previous
observation that not every residue energetically
involved in the CTD interface was contained within
CAC1 [23], the comparable affinity of the peptide–
CTD complex and the CTD–CTD dimer was unex-
pected. However, later studies have provided a possible
explanation for this observation. As in the CAC1 pep-
tide, a-helix 9 becomes only permanently structured
when the CTD monomers dimerize [25,26], and then a
substantial entropy penalty must be paid on the forma-
tion of both the peptide–CTD and the CTD–CTD
complexes [6]. As expected, CAC1 is able to efficienty
inhibit the in vitro assembly of the HIV-1 capsid
(R. Bocanegra and M. G. Mateu, unpublished results).
To map the region of CTD where CAC1 actually

binds, 2D-NMR titration experiments were attempted
but, because of the low solubility of the peptide, we
were unable to describe the interacting residues. We
have started the design of modified peptides of CAC1
with improved solubilities (in collaboration with
C. Cavasotto, Houston, TX, USA), and have been able
to map the region of CTD where a modified CAC1
peptide is bound, by using
1
H-
15
N- heteronuclear single
quantum coherence (HSQC) NMR experiments. The
CA small-molecule interactions J. L. Neira
6112 FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS
residues of dimeric CTD that experienced the largest
changes in chemical shifts, and ⁄ or their intensities were
significantly decreased in the presence of the peptide (in
a 2 : 1 peptide ⁄ protein molar concentration), were
Asp152, Arg154, Val165, Leu190, Gly206 and Thr210
(J. L. Neira, unpublished results). All of these residues
are involved in the dimerization interface of nonmutat-
ed CTD, or are located proximal to the interface (as
shown by the X-ray structure of CTD) [8]. Although it
has not been possible to ascertain the structure of
CAC1 upon binding to CTD by transfer NOE experi-
ments, it may be reasonable to assume (based on other
structural studies, see below) that it acquires an
a-helix-like conformation. We are currently developing
and designing new variants of CAC1 not only with

an improved solubility, but also with a larger helicity
to assess whether the latter improves the affinity for
CTD.
A phage display peptide as an inhibitor of CA
assembly
A CTD-binding, 12-mer peptide, CAI, was identified
by Stitch et al. [27] using phage display techniques.
The sequence of CAI is: ITFEDLLDYYGP, which
does not bear any resemblance to the sequence of the
dimerization helix of CTD (see above). CAI is the first
reported peptide able to inhibit the assembly of the
mature HIV-1 capsid in vitro. However, because of the
lack of permeability of cells to CAI, the peptide was
unable to inhibit HIV-1 infection ex vivo. The CAI
peptide isolated in solution is random-coil, but it
adopts an a-helical conformation upon binding to
CTD, as shown by the X-ray structure of the nonmu-
tated-CTD–CAI complex [28] (Fig. 2A, main panel).
The aromatic moiety of Phe3, together with Leu6 and
Ile1, are the residues of CAI with the largest number
of protein contacts (Fig. 2A, inset); these data suggest
that the CTD–CAI interaction occurs mainly through
hydrophobic contacts.
The X-ray structure of the CAI–CTD complex is a
five helix bundle [28] (Fig. 2A, main panel). In this
bundle, the dimerization helix of CTD (a-helix 9)
reorients to maximize interactions with CAI; this
movement results in a change of the buried surface at
the dimeric interface when compared with the buried
interface in the dimeric CTD. The affinity constant of

CAI for CTD was measured by mapping the changes
in
15
N-HSQC NMR spectra upon peptide addition
[27]; the dissociation constant is 15 ± 7 lm, which is
comparable to that of the CTD–CTD dimer, and
higher than that of CAC1. Similar to the results
obtained with CAC1 (see above), this value can be
explained because an entropy penalty must be paid
during folding of both the CAI peptide and a-helix 9
of CTD during formation of the peptide–
protein and protein–protein complexes.
These NMR experiments also allowed the determina-
tion of the CTD binding site. However, instead of using
the dimeric wild-type CTD species, Stitch et al. [27]
employed the monomeric CTD mutant (CTDW184A ⁄
M185A) in the titration experiments. Thus, they were
able to observe the chemical shifts of the whole dimer-
ization helix of CTD [27] (otherwise, those chemical
shifts are too broad to be observed because of the
presence of interconverting conformers) [29]. The bind-
ing site of CTD encompasses residues Tyr169 to
Val191, which agrees with the X-ray structure of the
nonmutated-CTD-CAI complex [28]; thus, the peptide
binds to a groove created by a-helices 8, 9 (the
dimerization helix) and 11 of CTD (Fig. 2A, main
panel) and, in addition, the kink at Thr188 in CTD is
less pronounced than in each of the monomers of the
dimeric CTD. Finally, it is important to note that some
of the residues of CTD bound to CAI are also involved

in binding to CAC1 and vice versa (see above); these
data suggest that the modes of binding of both peptides
are quite similar, if not identical.
Bartonova et al. [30] have recently shown that resi-
dues Tyr169, Asn183, Glu187 and Leu211, all of which
form the binding pocket site of CAI, are necessary for
the quaternary structure integrity of CTD [30]. When
residues Tyr169, Asn183, Glu187 and Leu 211 were
replaced with alanine, the resulting mutants were
competent for immature capsid-like particle assembly
in vitro and budding of immature-like capsids. The
results obtained by Bartonova et al. [30] pinpoint an
indirect (allosteric) effect of CAI binding on the assem-
bly of the immature capsid lattice, but a direct binding
effect in the conserved CTD binding pocket during
mature assembly. On the other hand, mutations at
Tyr169 and Leu211 do not yield mature-like particles
and give rise to non-infectious virions with nonregular
mature cores. The X-ray structures of these two
mutants differ from that of wild-type, and those of the
assembly-competent mutants E187A and N183A: the
conformations of the two assembly-incompetent
mutants (Y169A and L211A) in the absence of CAI
are the same as the structures of any mutant when
complexed to the peptide. Interestingly, Glu187
(together with Ser178, Glu180 and Gln192) is one of
the residues that decreases the association constant of
CTD, as previously demonstrated in an alanine
scanning of the dimerization interface [23].
Barklis et al. [31] have shown that CAI does not

block Gag assembly [27], but dismantles HIV-1 CA
J. L. Neira CA small-molecule interactions
FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS 6113
tubes [31]. The authors suggest two possible explana-
tions: (a) CAI modifies the CTD dimer interface (as
suggested based on the X-ray structure of the different
complexes [28,30]) and (b) CAI is able to replace the
a-helix 4 of NTD, which binds to a CTD groove, and
which helps align NTDs and CTDs around the CA
hexamers [6]. Thus, binding of CAI to CTD would
weaken the hexamers, impair assembly and destabilize
the assembled cores. In that way, CAI would act dur-
ing assembly and uncoating of the tubes. In summary,
CAI is the first peptide that has been shown to
efficiently inhibit CA assembly.
Variants of CAI peptide
Armed with this structural information on CAI, Zhang
et al. [32] used a structure-based rational design
approach to stabilize the a-helical structure of CAI
and also to convert it into a cell-penetrating peptide.
The resulting peptide, NYAD-1, contains a covalent
bridge between two amino acids separated four resi-
dues away; this bridge (the so-called ‘hydrocarbon sta-
ple’) acts by restricting the conformational freedom of
CAI. The sequence of NYAD-1 is: ITFXDLLXYY
GKKK, where X is the nonstandard amino acid (S)-2-
A
B
C
Fig. 2. The complexes of CTD with different

peptides and organic molecules. (A) Main
panel: structure of CTD (green) in complex
with the peptide CAI (purple) (accession no.
2buo) [28,30]; the helices are named after
the elements of structure in the wild-type
CTD in Fig. 1. Inset: the interface region is
shown with residues Phe3 (CAI) and Glu180
and Asn183 (CTD) indicated (the labels are
in different colours: blue for protein residues
and black for the peptide amino acids); for
the sake of clarity, only these two residues
of the CTD are shown. (B) Main panel:
structure of CTD (green) in complex with
the peptide NYAD-1 (cyan) (accession no.
2k1c) [33]; the helices are named after the
elements of structure in the wild-type CTD
in Fig. 1. Inset: the interface region is
shown with residues Phe3 (CAI) and
Asn183 (CTD) indicated (the labels are in dif-
ferent colours); for clarity, only Asn183 is
shown. (C) The structure of NTD in complex
with the small organic molecule CAP-1
(accession no. 2pxr) [36]; the helices are in
red, the b-sheets in yellow and the loops in
green. The circle at the bottom of the figure
indicates the region where the binding of
CAP-1 occurs. The figures were produced
using
PYMOL () [39].
CA small-molecule interactions J. L. Neira

6114 FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS
(2¢-pentenyl) alanine, which works as the ‘staple’
between both residues of the peptide; that staple makes
CAI more helical.
The NYAD-1 peptide is able to penetrate cells and
disrupts the assembly of both immature- and mature-
like virus particles in cell-free and cell-based systems
in vitro [32]. The NYAD-1 peptide demonstrates
enhanced helicity when isolated in solution, which
appears to suggest that the higher the helicity, the
higher the inhibition of assembly. The affinity of
NAYD-1 for CTD is the largest reported to date, with
values close to 1 lm. The difference in affinity from
the original wild-type CAI (approximately 15 lm; see
above) and with the CTD domain itself can be
explained by the higher helicity of the isolated peptide
(i.e. the NYAD-1 peptide, unlike CAC1, CAI and even
the CTD monomer, does not have to pay such a large
entropy penalty during binding to CTD because it is
already helically structured).
Zhang et al. [32] have also shown by using
15
N-
HSQC-NMR that the binding site of NAYD-1 to the
protein encompasses residues Phe169 to Val191, desta-
bilizing rather than dissociating the nonmutated
dimeric CTD (i.e. NYAD-1 does not disrupt the
dimeric structure of the wild-type CTD). The residues
observed in the binding region are the same as in CAI
(see above). Furthermore, Zhang et al. [32] have solved

the solution structure of the monomeric double mutant
CTDW184A ⁄ M185A in complex with NYAD-1
(Fig. 2B, main panel). In the structure, the a-helix 9 is
fully formed, with the kink present at the Thr188 [33],
in contrast to what happens with the structure of the
isolated monomeric double mutant [34]; thus, NYAD-
1 makes the dimerization helix of CTD adopt a more
native-like structure (if we assume native-like structure
to comprise that shown by each of the monomers in
the wild-type dimeric CTD). The peptide binds to a
hydrophobic pocket formed by residues of the four
helices, and it adopts a helical conformation (as CAI)
where its hydrophobic side chains (especially Phe3)
make extensive contacts with specific hydrophobic
patches of the double monomeric mutant (Fig. 2B,
inset). When a comparison of the NMR structure of
the complex NYAD-1-CTDW184A ⁄ M185A with the
X-ray structure of CAI-CTDW184A ⁄ M185A is carried
out, the differences are mostly observed in the
movement of a-helix 9 (the dimerization helix in the
wild-type nonmutated CTD); this helix is pushed away
much further from its original wild-type position in the
design of Stitch et al. [27] (6 A
˚
) than in that of
Zhang et al. [32] (3 A
˚
). It is tempting to suggest that
this short movement is also responsible for the larger
affinity constant of NYAD-1. Although the ability of

NYAD-1 to dismantle HIV-1 CA tubes has not been
tested, by analogy to the CAI peptide [31], it is likely
to have similar activity.
In summary, NYAD-1, a variant of CAI, is the first
peptide able to interfere with CA interactions during
HIV-1 assembly (both mature- and immature-like virus
particles), penetrate cells and inhibit the infectivity of
HIV-1.
Small organic molecules as inhibitors
of CTD self-assembly
The first breakthrough in identifying small-molecule
inhibitors of HIV-1 assembly was reported by Tang
et al. [35], with molecules not designed specifically
against CTD, but rather the whole CA. The first mole-
cule, N-(3-chloro-4-methylphenyl)-N¢-{2-[({5-[(dimeth-
ylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea)
(CAP-1) (Scheme 1), has dose-dependent inhibition in
viral infectivity assays, and its affinity for the NTD of
CA is approximately 800 lm. Both X-ray and NMR
show that CAP-1 binds to the full-length CA, in a
pocket formed at the point where helices 1, 2, 4 and 7
of the NTD interact [36] (interestingly, this region is
spatially close to the groove created by a-helices 8 and
9 of CTD, where CAI binds, see above) (Fig. 2C). The
protein undergoes a remarkable conformational change
as CAP-1 binds with the aromatic residue of Phe32,
which is moved away from its buried position in the
protein. This movement creates a large cavity where the
aromatic ring of CAP-1 inserts; the urea and dimethyla-
monium groups of CAP-1 also make contacts with the

side chains and ⁄ or backbones of several residues in the
protein. Thus, most of the contacts of CAP-1 with CTD
are hydrophobic, similar to the peptides (see above).
Scheme 1. The CAP-1 and CAP-2 comp-
unds designed by Tang et al. [35] and Kelly
et al. [36].
J. L. Neira CA small-molecule interactions
FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS 6115
Helices 4 and 7 of NTD form the interface required
to create a stable hexagonal CA lattice of the virion
capsid [37,38], and these helices pack against a-helices
8, 9 (the dimerization helix) and 11 of the CTD
domain. Thus, CAP-1 does not inhibit capsid assem-
bly through direct binding to CTD but rather because
it hampers the proper docking of both NTD and
CTD.
A second organic compound, CAP-2 (Scheme 1),
was shown also to bind the NTD of CA, but it is
cytotoxic and it was not investigated further [35].
Conclusions
In summary, it appears that even closely designed pep-
tides (such as CAI and NYAD-1) display subtle, but
key structural differences that account for the different
inhibition properties exhibited in vitro and in vivo.Itis
also clear that there are multiple ways to disrupt (or
better destabilize) the dimeric CTD because all the
peptide designs suggest slightly different modes of
binding. These modes of binding appear to be related
to: (a) the high flexibility of the a-helix-8- a-helix-9
interface (Fig. 1) and (b) a high number of hydropho-

bic contacts through the aromatic moieties of the pep-
tides (the hydrophobic contribution also appears to be
important in binding of CAP-1). Rather than targeting
dimerization of the CTD directly, studies with this
molecule demonstrate that another effective strategy
for disrupting the assembly of the virion capsid
involves targeting the whole CA protein and regions
close to the CTD. Thus, these peptides and small
organic molecules, together with the structures of the
complexes that they form with CA, provide an impor-
tant basis for the design of new anti-HIV-1 agents
aimed at inhibiting capsid assembly in vivo.
Acknowledgements
Both referees are thanked for their helpful suggestions
and discussion. This work was supported by grants
from Ministerio de Sanidad y Consumo (FIS 01⁄
0004-02), Ministerio Ciencia e Innovacio
´
n (SAF
2008-05742-C02-01; CSD2008-00005), FIPSE private
Foundation (Exp: 36557⁄ 06) and Generalitat Valenci-
ana (ACOMP ⁄ 2009⁄ 185). Drs Mauricio G. Mateu and
Francisco N. Barrera are thanked for critically reading
the manuscript and for their collaboration over all
these years. Dr Anjali P. Mascarenhas and Professor
Karin Musier-Forsyth are thanked for critically read-
ing and correcting an early version of the manuscript.
Marı
´
a del Carmen Lidon-Moya and Estefanı

´
a Hurtado-
Go
´
mez are acknowledged for their careful experimental
work. May Garcı
´
a, Marı
´
a del Carmen Fuster, Javier
Casanova and Olga Ruiz de los Pan
˜
os are deeply
thanked for providing excellent technical assistance.
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