Enhanced Replication of R5 HIV-1 Isolates invitro by a Small-Molecule Reagent Targeting HIV-1 Protease

Full text of DOI:10.1002/cmdc.201300051

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DOI: 10.1002/cmdc.201300051
Enhanced Replication of R5 HIV-1 Isolates in vitro by
a Small-Molecule Reagent Targeting HIV-1 Protease
Nick J. Wardle,*[a] Harry R. Hudson,[b] Ray W. Matthews,[b] Christine M. Nunn,[b]
Cherelyn Vella,[b] and S. W. Annie Bligh[c]
Entry of human immunodeficiency virus (HIV) into host cells re-
quires the initial attachment of virion envelope glycoprotein
gp120 to cell-surface receptor CD4, and then interaction with
chemokine receptor CXCR4 (expressed on T-lymphocytes) or
CCR5 (expressed on monocytes/macrophages).[1] The chemo-
kine-receptor subtype exploited for cell entry determines the
tropism of HIV-1 strain involved;[2] those utilising CXCR4 are
termed T-tropic X4 viruses, and those exploiting CCR5 are M-
tropic R5 viruses. Dual-tropic R5X4 viruses are also known. R5
viruses are important in viral transmission,[3] being responsible
for 95% of new infections, and are the predominant species
replicating during asymptomatic stages of the disease. There-
after, evolutionary emergence of X4 viruses characterises signif-
icant disease progression and the advent of symptomatic
stages.[4] Hence use of clinically relevant R5 viruses in vaccine
and drug development is important, motivating interest in ad-
dressing the practicalities of achieving R5 virus titres suitable
for in vitro biological studies. In HIV replication assays, mono-
cyte-derived macrophages (MDMs) are likely to be more phys-
iologically representative than continuous cell lines.[5] CD8 T-
cell-depleted, cytokine-stimulated peripheral blood mononu-
clear cells (PBMCs) are routinely used for R5 replication studies,
containing high levels of activated memory cells (CD45RO+),
which are permissive to R5 viruses.[6] However, R5 replication in
stimulated PBMCs is slow, and the virus titres obtained are usu-
ally poor.[7] Hence scope exists for further development of in
vitro R5 virus replication models.
est as potentially susceptible to acid-catalysed intramolecular
(5-exo-P-tet) transesterification in the environment of the pro-
tease active site. The O-alkyl propylphostonate[11] function so
formed would be highly activated to nucleophilic displacement
at phosphorus, and reactive, therefore, towards critical enzy-
matic residues and structural features. “Molegro” virtual dock-
ing experiments[12] indicated docking of 1 within the protease
active site, identifying H-bonding interactions involving P=O
(O!H interatomic distance 2.6 ꢀ) and hydroxy functions, re-
spectively, with Asp25 residues of both monomers of the
enzyme (Figure 1). These interactions could themselves inhibit
protease activity, however, proximity of the phosphonate func-
Described here are the unexpected results of HIV replication
studies using this model of bioevaluation in our investigation
of structures targeting HIV-1 protease (HIV-1 PR). The original
objective was to present electrophilic phosphonate functions
in putative substrates of HIV-1 PR,[8,9] developed in pursuit of
a viable mechanism-based approach[10] to inhibition. In this
context, the g-hydroxyphosphonate arrangement was of inter-
Figure 1. Lowest docked energy conformation of compound 1 in the active
site binding pocket of HIV-1 PR. Residues of the second monomer of HIV-
1 PR are given prime notation. Inset: structure of compound 1.
[a] Dr. N. J. Wardle
Institute for Health Research and Policy
London Metropolitan University
166–220 Holloway Rd, London N7 8DB (UK)
E-mail: n.wardle@londonmet.ac.uk
tion of 1 to enzymatic Ile50/50’ N atoms (P!N interatomic
distances 6.6 ꢀ and 4.7 ꢀ, respectively) also suggested scope
for hydrolytic sequestration of a Ile50/50’-bound structural
water molecule critical to enzyme–substrate binding interac-
tions.[9,13]
[b] Prof. H. R. Hudson, Prof. R. W. Matthews, Dr. C. M. Nunn, Dr. C. Vella
Faculty of Life Sciences and Computing
London Metropolitan University
166–220 Holloway Rd, London N7 8DB (UK)
[c] Prof. S. W. A. Bligh
The azacycloalkyl scaffold of 1 was developed from malo-
nate 2[14] via phase-transfer-catalysed alkylation. This employed
1,3-dibromopropane in a heterogeneous toluene/potassium
carbonate system with 18-crown-6 and Aliquot 336 catalysts
acting synergistically (Scheme 1).[15] Essential for successful
Department of Complementary Medicine
School of Life Sciences, University of Westminster
115 New Cavendish St., London W1W 6UW (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300051.
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Table 1. Compound 1 enhances replication of R5 HIV isolates in peripher-
al blood mononuclear cells (PBMCs).
Virus[a]
Fold change[b]
Virus[a]
Fold change[b]
PE124
010
031
11.5ꢀ0.39
10.4ꢀ0.19
6.7ꢀ0.16
9.1ꢀ0.28
041
048
094
114
9.7ꢀ0.43
12.6ꢀ0.60
9.2ꢀ0.86
6.9ꢀ0.10
037
[a] For details, see Ref. [19] (PE124) or Ref. [20] (all other viruses); [b] Fold
change in virus yield; for details, see Ref. [21]. Data represent the mean
ꢀ SEM of three or more independent experiments.
Gosselies, Belgium). Compound 1 was tested for functional ac-
tivity towards human CCR5 receptor using an aequorin assay[24]
for GPCR-mediated Ca2+ signalling, and it was also tested in
Scheme 1. Reagents and conditions: a) Br(CH2)3Br (1.5 equiv), K2CO3 (3 equiv),
18C6/Aliquot 336 (cat), PhMe, 95–1008C, 24 h, 26%; b) LiBH4–MeOH
(4 equiv), Et2O, N2, 08C!RT, 1 h, then D, 2 h, 25%; c) PivCl (1 equiv), N-meth-
ylmorpholine, CH2Cl2, N2, 08C, 5 min, then RT, 72 h, 29%.
a
[
125I]-macrophage inflammatory protein (MIP)-1b radioli-
gand[25] binding competition assay.
While no change in activity was observed in the aequorin
assay at a compound concentration of 50 mm, compound
1 gave rise to a modest (21%) enhancement in radioligand
binding at this concentration. These results are consistent with
the concept that 1 binds CCR5 at an alternative site to that of
chemokines, causing a conformational change in the receptor
by an allosteric mechanism. Noncompetitive, allosteric modes
of interaction have been proposed for a number of low-molec-
ular weight CCR5 antagonists.[26] However, in light of the spe-
cificity of action of 1 towards R5-type viruses over their X4
counterparts, enhanced rates of HIV replication without promo-
tion of intracellular Ca2+ release implies facilitation of HIV cell
entry without modulation of CCR5 expression or transcriptional
activity. In the physiological situation, HIV gp120 competes
with chemokines for binding at receptor CCR5 (and CXCR4).[27]
Hence, potentiation of CCR5 binding of chemokine MIP-1b in-
dicates a capacity for reagent 1 to facilitate HIV gp120 binding
in macrophages cultivated in vitro in the absence of chemo-
kines. To our knowledge, this constitutes the first instance of
a small molecule potentiating HIV replication in this way.
These unexpected results are significant with respect to fa-
cilitating in vitro biological studies with the R5 virus group.
Monocytic cell line Mono Mac 1 is now regarded as a viable
model for R5 HIV-1 infection,[5,28] and human acute monocytic
leukaemia cell line THP-1 offers qualified utility as such.[5] How-
ever, these systems are seen as complementary to physiologi-
cally representative infection models relying on MDMs. As cur-
rent methods produce poor R5 replication profiles in PBMCs,[6]
a reagent enhancing replication in this system would facilitate
the use of clinically relevant isolates in biological studies.
one-step cyclisation is the gradual introduction of base to
liquid phase, eliciting initial deprotonation and alkylation at
malonate carbon Cb. Kinetic preference for 6-exo-tet cyclisation
over both O-alkylation (8-exo-tet annulation) and intermolecu-
lar processes then directs site-selectivity to favour intramolecu-
lar, amide N-alkylation, generating the azacyclohexyl structure.
Slow, concomitant phosphonate dealkylation limited reaction
times, the optimum conditions being 24 hours at 95–1008C
(26% yield).
Chemoselective reduction of a non-enolisable malonate
function in the presence of amide required a lithium borohy-
dride/methanol system[16] (use caution for this protocol) in
order to generate diol 4 (15–25% yield). Steric factors rendered
the malonate function of 3 more refractory to reduction than
anticipated, even with lithium borohydride in large excess and,
along with the slow development of an unidentified polar by-
product, this limited reaction times. Derivatisation of one hy-
droxy function to pivaloate then generated 1 in 29% yield
over 3 days.
HIV-1 PR binding studies conducted on 1 indicated moder-
ate inhibitory activity towards the protease (IC50 =1.15 mm).
Meanwhile, the effect of compound 1 on HIV-1 replication was
examined in vitro at a compound concentration of 50 mm
using a panel of clinically relevant primary isolates of HIV-1 in
primary cell (CD8-depleted PBMC) cultures.[7,17,18] Unexpectedly,
virus replication was significantly enhanced in R5 isolates by
factors ranging from 6.7- to 12.6-fold (Table 1), as determined
by measurement of virus core protein p24 (determined using
a p24 ELISA; supplied by Innogenetics NV, Gent, Belgium).
Little or no enhancement was seen with the single X4 virus
Supporting Information
[19]
(HIV-1PE106
)
tested (data not shown).
Chemical and biological experimental protocols and details of the
computational methods used are given in the Supporting Informa-
tion.
The selective action of 1 towards R5 viruses over their X4
counterparts suggested interaction with the CCR5 receptor as
a potential cause. Compound 1 also contains a 1-arylcarbamo-
yl-piperidyl motif common to some therapeutically relevant
CCR5 ligands,[22] although other critical pharmacophores are
absent.[23] Hence, further investigation of this putative mode of
action was undertaken (assays performed by Euroscreen S.A.,
Keywords: antiviral agents · CCR5 · chemokines · HIV-1 · R5
isolates · viral replication · vaccine and drug development
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Received: February 4, 2013
Published online on March 18, 2013
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