capsid proteins of enveloped viruses as antiviral drug targets

Capsid proteins of enveloped viruses as antiviral drug targetsKlaus Klumpp1 and Thibaut Crepin2

Available online at www.sciencedirect.com

ScienceDirect

Viral proteins have enabled the design of selective and

efficacious treatments for viral diseases. While focus in this

area has been on viral enzymes, it appears that multifunctional

viral proteins may be even more susceptible to small molecule

interference. As exemplified by HIV capsid, small molecule

inhibitors can bind to multiple binding sites on the capsid

protein and induce or prevent protein interactions and

conformational changes. Resistance selection is complicated

by the fact that the capsid proteins have to engage in different

protein interactions at different times of the life cycle. Viral

capsid assembly and disassembly have therefore emerged as

highly sensitive processes that could deliver a new generation

of antiviral agents across viral diseases.

Addresses1 Novira Therapeutics, Inc., 3805 Old Easton Road, Doylestown, PA

18902, United States2 University of Grenoble Alpes-EMBL-CNRS, Unit for Virus Host-Cell

Interactions, 6 rue Jules Horowitz, 38042, France

Corresponding author: Klumpp, Klaus

([email protected], [email protected]) and

Current Opinion in Virology 2014, 5:63–71

This review comes from a themed issue on Virus structure andfunction

Edited by Wah Chiu and Thibaut Crepin

1879-6257/$ – see front matter, # 2014 Elsevier B.V. All rights

reserved.

http://dx.doi.org/10.1016/j.coviro.2014.02.002

IntroductionViral diseases are the source of significant morbidity and

mortality worldwide [1]. More than 20 virus families

contain known human pathogens. The development of

prophylactic vaccines and efficacious antiviral treatments

has been very successful against a few of these pathogens,

but large medical needs remain un-addressed and major

challenges remain even in those diseases for which treat-

ments are available. For example, the current treatment

options for HIV or Hepatitis B infections have had a major

impact on survival of chronically infected patients.

Optimal treatment can significantly delay the onset of

severe disease, but ongoing low level virus replication,

safety and tolerability issues and the difficulty to maintain

adherent to life-long daily drug administration are main

causes of treatment failure. Chronic inflammation, long

term drug administration and complexities associated

with polypharmacy and drug–drug interactions in many

www.sciencedirect.com

patients present additional health issues that need to be

addressed ([2–9]; http://aidsinfo.nih.gov/guidelines). It is

also of concern that only very few options are available to

manage severe acute infections that are caused by a

number of different viruses. Treatments are needed that

provide very fast onset of antiviral activity and highly

efficient shut-down of viral replication in these cases [9–11]. There is therefore an urgent need for additional

classes of antiviral agents to address serious unmet

medical need across a range of viral diseases.

Viral core or capsid proteins are emerging as interesting

targets for the development of new potent antiviral

agents. The process of encapsidation of viral nucleic acid

to enable the formation of virions and the infection of new

cells is complicated by the fact that the capsid forming

protein subunits have to interact with each other, and

with other viral and host proteins to form the capsid at the

right time during the viral life cycle and at the right

location in the infected cell. The capsid has to be stable

to protect the genome in extracellular environments, but

not too stable as to prevent efficient genome release after

entry into new host cells. The typical icosahedral or

conical capsid structures are formed from hexameric

and pentameric building blocks that emerge from slightly

different interaction of the same capsid protein subunits.

The capsid proteins have therefore evolved to be con-

formationally flexible to allow different functional inter-

actions with themselves (to form hexamers or pentamers)

and with other proteins. Even small interferences with

the ability of the capsid forming proteins to undergo

required conformational changes or changes in the

stability of protein interactions can disrupt critical steps

in the process between genome encapsidation and

release. In addition, many capsid forming proteins are

performing additional regulatory functions in infected

cells, such as modulation of host gene expression and

interference with immune recognition. Not surprisingly

therefore, the sequences of capsid proteins show high

sequence conservation levels and many single point

mutations are associated with replication deficiency.

Recent studies have described the identification of struc-

turally diverse small molecule inhibitors of viral replica-

tion that target these sensitive processes of genome

encapsidation and release. This short review will focus

on examples of inhibitors of HIV replication, which

exemplify the principle that targeting capsid forming

proteins with small molecules is feasible and can result

in different biological phenotypes, depending which step

in the encapsidation–release–host factor interaction net-

work is primarily inhibited. The structural flexibility of

capsid proteins allows the binding of small molecules to


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http://dx.doi.org/10.1016/j.coviro.2014.02.002

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64 Virus structure and function

different binding sites and substantial differences in

biological phenotype can be observed even for com-

pounds that bind to the same binding site on the protein.

HIV capsid protein is structurally flexibleThe HIV capsid (CA) protein is structurally flexible and

creates multiple different protein–protein interaction

surfaces with itself, other viral and host proteins at differ-

ent times during the viral life cycle [12�,13�]. CA is

synthesized as part of the Gag polyprotein and consists

of two independently folded domains, the N-terminal

domain (CA-NTD) and the C-terminal domain (CA-

CTD). CA is the main driver of Gag oligomerization in

the formation of the immature capsid, and CA forms the

mature, cone-shaped capsid after it is released from Gag

by proteolytic processing in the virion [12�,13�,14]. Drug

discovery for this target is now greatly facilitated by

structural information that has been generated using

native HIV cores and a variety of different protein con-

structs and methods, including cryo-EM, NMR and crys-

tallography [15��,16��,17–21]. The structural information

and molecular capsid models suggest very different

protein–protein interactions of CA in the immature

spherical capsid, as compared to the mature cone-shaped

capsid [12�,13�]. To ability of CA to undergo such

dramatically different protein interactions forming two

different types of hexameric building blocks is facilitated

by overall weak interactions between CA dimer

(Kd � 10–20 mM) and CA hexamers, and by the modu-

lation of CA interactions through other Gag protein

domains, especially SP1, which change after cleavage

by the HIV protease. Mutational analyses are consistent

with the structural models. Importantly, most single point

mutations result in replication deficiency, consistent with

the critical role that most amino acids play to maintain the

ability of the CA protein to adapt a number of different

conformations and protein–protein interactions through-

out the viral life cycle [22,23,24,25�].

HIV replication inhibitors targeting capsidCAP-1, BD, BM

A number of independent small molecule screens have

been performed, most of them looking for compounds

that could interfere with capsid assembly in vitro. The key

compounds identified in this way are summarized in

Table 1, and many of them have also been included in

another recent review on this topic [24]. Figure 1 shows

three major and well separated binding sites of small

molecule inhibitors targeting the HIV CA-NTD. The

first small molecule binding site on HIV CA was ident-

ified from a NMR screen. The screen identified two

compounds that bound to CA-NTD and could inhibit

salt induced CA aggregation in vitro. One compound was

toxic, while the other compound, named CAP-1, inhib-

ited HIV replication in cell culture at a concentration of

100 mM. 1H–15N HSQC NMR and crystallography data

were consistent with binding of CAP-1 to an induced


hydrophobic pocket at the junction of 5 a-helices at the

base of the CA-NTD domain [26,27] (Table 1, Figure 1,

binding site highlighted in purple). This discovery pro-

vided the first indication that small molecule inhibition of

HIV capsid function was possible. It encouraged further

drug discovery efforts, even though the two pioneer

compounds did not bind to the site that had been targeted

by the in silico docking method, and despite the fact that

one was too toxic to use in antiviral assays, while the other

was so weakly binding to CA-NTD, that density for the

compound was not visible in the crystals. From a number

of efforts in different groups, the most potent compounds

to date that bind to the CAP-1 binding site came from

research performed at Boehringer Ingelheim. Using a

fluorescence based assembly assay with CA-NC fusion

protein, a number of different series of HIV replication

inhibitors were identified. Two series, named the BD

(benzodiazepine) and the BM (benzimidazole) series,

were further investigated and delivered compounds with

mean antiviral potencies of 70 and 62 nM, respectively

[28,29,30�] (Table 1). The binding of compounds from

these series to the CAP-1 binding site on the HIV CA-

NTD was determined by NMR and crystallography, and

binding affinity determined by NMR and ITC. The

binding of compound BD3 is shown in Figure 1 (purple

color) and indicates a different binding mode as compared

to the CAP-1 compound (compare top left zoom view,

CAP-1, to the top right, BD3). Compounds from the BD

series inhibited virion release from HIV producing cells,

consistent with the inhibition of immature capsid assem-

bly. In contrast, the BM series allowed virus budding, but

prevented capsid maturation. Interestingly, the biological

phenotype of the two series was strikingly different,

despite of the fact that both were binding to the same

binding site [30�]. Virus passaging resulted in the selec-

tion of different resistance mutations for both series. The

selected mutations were consistent with important inter-

actions of the compounds in the binding site. Most

mutations significantly reduced HIV replication capacity

[30�].

PF-74

A different binding site was identified by researchers at

Pfizer based on a hit from a phenotypic cell based antiviral

assay screen. In this case, the antiviral target was first

identified by virus passaging and resistance selection in

cell culture, which resulted in the selection of a resistant

virus variant with a T107N mutation in the CA-NTD

coding sequence. The prototype compound from this

series is PF-74 (PF-3450074) with a mean antiviral

EC50 of 570 nM [31��] (Table 1). Crystallography con-

firmed the binding of PF-74 to a new, pre-existing site on

the CA-NTD domain. This binding site is shown in

Figure 1 with red highlight. Binding of PF-74 to this site

did not induce any apparent conformational change on

the NTD domain. Interestingly, PF-74 accelerated CA

assembly in vitro, in contrast to the compounds that bind


Capsid proteins as antiviral drug targets Klumpp and Crepin 65

Table 1

Structures and biological activities of selected antiviral compounds that interfere with viral capsid assembly

Compound Structure Activity Reference

CAP-1 CA-NTD Kd � 800 mM

EC95 � 100 mM

CC50 > 100 mM

[26]

BD 1

EC50 = 70 � 30 nM (n = 21)

CC50 > 28 mM

[30�]

BD 3

EC50 = 480 nM [30�]

BM 1

EC50 = 62 � 23 nM (n = 53)

CC50 = 20 mM

[30�]

PF-74 EC50 = 570 � 260 nM

CC50 = 69 � 15 mM

[31��]

#6

Kd = 500 nM (ITC)

IC50 = 350 nM

EC50 = 950 nM

CC50 = 57 mM

[41]

www.sciencedirect.com Current Opinion in Virology 2014, 5:63–71


Table 1 (Continued )

Compound Structure Activity Reference

#4 [41]

#1 [42]

IC50 = 1200 nM [41]

[42]

BI-1 pyrrolo-pyrazolone Kd = 20 mM (ITC, NMR)

EC50 = 7.5 � 2.1 mM

CC50 > 91 mM

[43��]

BI-2 Kd = 3 mM (ITC, NMR)

EC50 = 1.4 � 0.66 mM

CC50 > 76 mM

[43��]

ST-148 EC50 (DENV-1) = 2.8 � 1.1 mM

EC50 (DENV-2) = 0.016 � 0.01 mM

EC50 (DENV-3) = 0.51 � 0.42 mM

EC50 (DENV-4) = 1.2 � 0.14 mM

CC50 > 50 mM

[44�]

Bay 41-4109

EC50 = 0.05 mM

CC50 = 7 mM

[50]

AT-130

EC50 = 0.13 mM

CC50 > 61 mM

[51]

HAP-1

EC50 = 0.36 � 0.05 mM [52]

Current Opinion in Virology 2014, 5:63–71 www.sciencedirect.com


Figure 1

90º90º

70º

Nt

Ct

Current Opinion in Virology

Three structurally defined inhibitor binding sites on the N-terminal domain of the HIV CA protein. The center panel shows two protomers of CA, one in

cartoon and one in surface representation, with the N-terminus (Nt) and C-terminus (Ct) indicated. Three small molecule binding sites are indicated by

purple (2JPR, 4E91), red (2XDE) and yellow (4E91) color highlights. The zoom-in views show the binding of representative compounds CAP-1 (purple,

top left [27]), BD3 (purple, top middle [30�]), PF-74 (red, bottom left [31��]) and #4 (yellow, bottom right [41]).

to the CAP-1 binding site, which inhibited CA assembly

in vitro. Another difference was the fact that PF-74

inhibited both early and late events in the HIV life cycle

with similar potency, while the inhibition of late events

(virion release or maturation) was driving the antiviral

potency of the compounds that bound to the CAP-1

binding site. Early inhibition of HIV replication by PF-

74 occurred before reverse transcription [31��]. Further

studies showed that PF-74 could bind to and destabilize

mature capsids, while capsids with the PF-74 resistance

mutation were not affected by incubation with the com-

pound [32]. CA mutations which stabilized the capsid

could confer resistance to PF-74, while capsids with

destabilizing mutations were more sensitive to PF-74

[32]. The binding site of PF-74 was separate from that

of CAP-1 and also different from the site where the host

factor cyclophilin A binds to CA. Interestingly, cyclos-

porine A, which inhibits cyclophilin A binding to CA, was

strongly antagonistic with PF-74 in the antiviral assay. In

addition, CA mutations that prevent cyclophilin A bind-

ing were also resistant to PF-74. Depletion of cyclophilin

A by siRNA treatment reduced HIV sensitivity to PF-74

[32]. The simultaneous presence of cyclophilin A and PF-


74 therefore increased the antiviral activity of PF-74,

consistent with the notion that cyclophilin A and PF-

74 can bind to CA simultaneously.

Depletion of another host factor, transportin SR2/

TNPO3, also decreased the sensitivity of HIV to inhi-

bition by PF-74. In this case, the cooperative effect was

likely due to the fact that both TNPO3 and PF-74 are

capsid destabilizing factors and the simultaneous pre-

sence of TNPO3 and PF-74 can accelerate premature

capsid disassembly [33]. The concept that premature

capsid disassembly is deleterious for HIV is also consist-

ent with recent data that suggest that the HIV capsid

remains intact in the cytoplasm and that uncoating may

happen at the nuclear pore [34,35]. Intact capsid has been

observed at the nuclear pore, capsid interaction with the

nuclear pore protein NUP358 is required for the peri-

nuclear localization of capsid, and NUP358 has been

identified as a required co-factor for HIV replication in

siRNA screens [34,35,36,37]. These results suggest that

HIV, like a number of other viruses, avoids exposure of

the viral RNA to RNA sensing antiviral host factors in the

cytoplasm of infected cells, or that maintenance of the



pre-integration complex in the context of the capsid may

increase reverse transcription efficiency.

CPSF6-358, a truncated cytosolic form of the protein

CPSF6 (cleavage and polyadenylation specific factor 6)

was also recently found to bind to the CA-NTD domain

and restrict HIV replication. Interestingly, the binding

site of CPSF6-358 on CA overlapped with that of PF-74,

and both molecules showed competitive binding to CA

[38]. Interestingly, different forms of cytosolic CPSF6

variants could either stabilize or destabilize capsid, in

both cases leading to replication inhibition [39,40].

Apex binding benzimidazoles

The Boehringer Ingelheim group also identified another

series of benzimidazoles from their in vitro capsid assem-

bly screen that, according to 1H–15N NMR, affected

different sets of residues as compared to the BM series

described above. In agreement with the NMR data, a co-

crystal structure showed binding of a representative com-

pound from this series to a pocket at the base of the

cyclophilin A binding loop on HIV CA. This binding site

is located at the apex of the CA-NTD helical bundle and

is well separated from the other two binding sites

described above [41,42] (Table 1, #1, #4, #6). The apex

binding site with compound #4 [41] is highlighted in

Figure 1 with yellow color. The compounds from this

original hit series showed preliminary SAR with Kd values

determined with CA-NTD by ITC between 0.5 and

43 mM, which correlated well with Kd values determined

from chemical shift changes by NMR. The compounds

inhibited capsid assembly in the oligonucleotide acti-

vated CA-NC assembly assay with IC50 values between

0.35 and 6.1 mM and the best compound had an antiviral

EC50 value of 0.95 mM. Although the binding site of this

series was close to the cyclophilin A binding site, both

could bind simultaneously to CA-NTD as determined by

NMR. In addition, compounds from the previously

described BD series could bind to the CAP-1 binding

site in the presence of compounds from this series,

consistent with the large separation of the two binding

sites (purple and yellow in Figure 1) [41]. There was no

significant conformational change induced by binding

according to crystallography. Assembly inhibition could

result from an interference of CA-NTD-mediated inter-

hexamer contacts that are required in the formation of the

mature capsid [16��,41]. Interestingly, the presence of a

compound from this series was found to improve crystal-

lization of CA-NTD and enabled the generation of tern-

ary co-crystal structures with other compounds binding to

the CAP-1 binding site that had failed to generate co-

crystal structures with CA-NTD on their own [42]. These

results provided further proof that the two compound

series could bind simultaneously to CA-NTD. The

improved crystallization performance was hypothesized

to be related to the facilitation of protein dimerization


mediated by the compound, facilitating nucleation of

crystallization.

Pyrrolo-pyrazolones BI-1/BI-2

Another interesting series of compounds targeting HIV-

CA were identified from a cell based, single cycle HIV

infection assay. The representative pyrrolo-pyrazolones

BI-1 and BI-2 inhibited HIV replication in single cycle

(EC50 = 8.2 (BI-1), 1.8 (BI-2) mM) and multi-cycle

(EC50 = 7.5 (BI-1), 1.4 (BI-2) mM) assays [43��] (Table

1). But these compounds did not inhibit infectious virus

formation in HIV producer cells (EC50 > 43 mM). The

target and binding site was determined by resistance

selection, ITC, NMR and crystallography as overlapping

with the PF-74 binding site on the CA-NTD domain (PF-

74 binding site highlighted in red in Figure 1). Similar to

PF-74, there was no apparent conformational change

associated with compound binding observed in crystal-

lography and the compounds were accelerators of CA-NC

assembly in vitro. There were, however, major phenoty-

pic differences between this series and PF-74. (a) PF74

inhibited reverse transcription and had a de-stabilizing

effect on capsid, while this series did not inhibit reverse

transcription and had a stabilizing effect on capsid, while

the concentration of 2-LTR circles was reduced, consist-

ent with an inhibition of nuclear import; (b) PF-74

inhibited early and late phases of HIV replication, while

this series only inhibited the early phase.

HIV capsid inhibitor summary

The characterization of antiviral compounds that inter-

fere with HIV capsid function indicates a striking multi-

tude of binding sites and phenotypic profiles. Three

clearly defined small molecule binding sites have been

identified on the N-terminal domain of CA-NTD alone.

In addition, it has been interesting to learn that the

binding of compounds to the same binding site on CA-

NTD could have very different biological consequences

in the HIV life cycle. These results are all consistent with

the multifunctional role of the capsid protein that

involves a number of different protein conformations

and interactions with different protein surfaces and host

factors. It will be highly interesting to learn more about

the potential to increase the antiviral potency by lead

optimization and by the combination of compounds bind-

ing to different binding sites on CA. In addition, the

barrier to resistance remains to be better understood,

which may also be affected by combination of compounds

binding to different binding sites. Finally, the principles

observed with HIV capsid targeting are likely to translate

into opportunities for targeting capsid proteins of other

enveloped viruses.

Capsid inhibitors demonstrate antiviralactivity in vivoThere is already ample evidence to suggest the translat-

ability of capsid inhibition principles learned in the HIV



field to other viral disease indications, and antiviral

activity has been demonstrated in animal models in a

number of cases. For example, a group at SIGA Tech-

nologies recently published the discovery of a small

molecule, ST-148, which targets Dengue virus capsid

protein, showed activity across all four Dengue serotypes

and reduced viremia in a non-lethal AG129 mouse model

[44�].

BAY-41-4109, a compound that interferes with HBV

capsid assembly has also shown evidence of in vivoantiviral activity in the HBV transgenic mouse model

and in Alb-uPA/SCID mice with humanized chimeric

liver [45,46]. Unfortunately, treatment in the humanized

mouse model was only for 5 days and started already 10

days after infection. These conditions did not provide

information on possible antiviral activity of the capsid

assembly inhibitor in a chronic HBV infection model.

Similar to the HIV case, compounds with potent antiviral

activity have been identified that accelerate rather than

inhibit HBV capsid assembly in vitro. A recently pub-

lished crystal structure of the HBV capsid obtained in the

presence of the HBV capsid assembly effector AT-130

from the phenylpropenamide class indicates that AT-130

binds to an overlapping binding site with HAP-1, a

compound from a different structural class of heteroar-

yldihydropyrimidines [47��]. HAP compounds were

shown to stabilize capsid protein dimer interactions

and to induce misassembly by changing the geometry

of these dimer interactions. In contrast, the primary

phenotype of phenylpropenamides is the block of viral

RNA packaging and the formation of empty capsids

without affecting capsid stability or geometry [48,49].

Similar to the HIV case, therefore, compound binding to

an overlapping binding site on the viral capsid protein can

result in substantially different biological effects.

ConclusionViral capsid proteins of enveloped viruses have emerged

as promising targets for the design of a new generation of

antiviral agents. It has become clear that the processes of

viral genome encapsidation and release that are depend-

ent on controlled capsid assembly and disassembly are

highly sensitive to even subtle molecular disturbances

that increase or decrease capsid stability, increase or

decrease the rate of capsid assembly, change the geo-

metry or conformation of capsid building blocks or inter-

fere with host factor engagement. A number of different

hit series of small molecule inhibitors of viral replication

targeting these processes have already been identified

from biochemical and cell based assay screens. Although

most of these compounds are early stage tool compounds

or hits, they are important as they have demonstrated the

potential for multiple compound binding sites on capsid

proteins and the potential for multiple mechanistic phe-

notypes of inhibition. The biological requirement for

viral capsid proteins to be structurally flexible for the


engagement into very different molecular interactions at

different phases of the virus life cycle represents a viral

weakness and offers clear opportunities for pharmaco-

logic interference.

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capsid proteins of enveloped viruses as antiviral drug targets

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