Pentacycloundecane derived hydroxy acid peptides: A new class of irreversible non-scissile ether bridged type isoster as potential HIV-1 wild type C-SA protease inhibitors

Article abstract of DOI:10.1016/j.bioorg.2011.08.002

Novel peptides incorporating the PCU derived hydroxy acid (5-hydroxy-4-oxahexacyclo[5.4.1.0^2,6.0^3,10.0 ^5,9.0^8,11]dodecane) were synthesized and their activity against the resistance-prone wild type C-South African (C-SA) HIV-protease is reported. The attachment of peptides and peptoids to the PCU derived hydroxy acid resulted in a series of structurally diverse promising HIV-1 protease inhibitors. Amongst the nine novel compounds, 16, 17, 20 and 23 gave IC ₅₀ values ranging from 0.6 to 5.0 μM against the wild type C-SA HIV-1 protease enzyme. Docking studies and molecular dynamic (MD) simulations have been carried out in order to understand the binding mode of the PCU moiety at the active site of the HIV protease enzyme. A conserved hydrogen bonding pattern between the PCU derived hydroxy ether and the active site residues, ASP25/ASP25′, was observed in all active compounds.

Full text of DOI:10.1016/j.bioorg.2011.08.002

Bioorganic Chemistry 40 (2012) 19–29
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Pentacycloundecane derived hydroxy acid peptides: A new class of irreversible
non-scissile ether bridged type isoster as potential HIV-1 wild type C-SA
protease inhibitors
Rajshekhar Karpoormath a, Yasien Sayed c, Patrick Govender b, Thavendran Govender d,
Hendrik G. Kruger a, Mahmoud E.S. Soliman e, , Glenn E.M. Maguire a,
⇑
⇑
a School of Chemistry, University of KwaZulu-Natal, Durban 4001, South Africa
b School of Biochemistry, University of KwaZulu-Natal, Durban 4001, South Africa
c Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Wits 2050, South Africa
d School of Pharmacy and Pharmacology, University of KwaZulu-Natal, Durban 4001, South Africa
e Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
a r t i c l e i n f o
a b s t r a c t
Novel peptides incorporating the PCU derived hydroxy acid (5-hydroxy-4-oxahexacyclo[5.4.1.02,6
.
Article history:
Received 10 April 2011
Available online 16 August 2011
03,10.05,9.08,11]dodecane) were synthesized and their activity against the resistance-prone wild type C-
South African (C-SA) HIV-protease is reported. The attachment of peptides and peptoids to the PCU
derived hydroxy acid resulted in a series of structurally diverse promising HIV-1 protease inhibitors.
Keywords:
Amongst the nine novel compounds,16, 17, 20 and 23 gave IC50 values ranging from 0.6 to 5.0 lM against
Transition state analogs
5-Hydrozxy-4-oxahexacyclo
[5.4.1.02,6.03,10.05,9.08,11]dodecane peptides
HIV-1 wild type C-SA protease
PCU derived peptides
the wild type C-SA HIV-1 protease enzyme. Docking studies and molecular dynamic (MD) simulations
have been carried out in order to understand the binding mode of the PCU moiety at the active site of
the HIV protease enzyme. A conserved hydrogen bonding pattern between the PCU derived hydroxy ether
and the active site residues, ASP25/ASP250, was observed in all active compounds.
Ó 2011 Elsevier Inc. All rights reserved.
HIV protease inhibitor
Inhibitory concentration (IC50
Docking
)
Molecular dynamics
1. Introduction
the clinical efficacy of these agents [9,10]. Thus, there is a
continuing demand for newer mutant-resistant HIV–PR inhibitors.
HIV is a global health problem and has become a major threat
to the existence of the human race. It has already caused an esti-
mated 25 million deaths worldwide and has generated profound
demographic changes in the most heavily affected countries. In
2007 alone there were 2.7 million new HIV infections and 2 mil-
lion HIV-related deaths worldwide [1]. The human immunodefi-
ciency virus (HIV) encodes an aspartic protease (PR) that is
essential for the formation of mature and infectious virions
[2,3]. The HIV–PR is an established target in the chemotherapy
of acquired immunodeficiency syndrome (AIDS) and intensive
efforts have been directed to develop potent, orally available,
peptidomimetic inhibitors for this enzyme [3–6]. At present, nine
PR inhibitors have been approved by the FDA, and several others
are now in clinical trials [6–8]. However, long-term anti-retrovi-
ral therapy for HIV infected patients promotes the emergence of
resistance mutations of the HIV–PR, and consequently reduces
Fortunately, HIV–PR variants expressing resistance to inhibitors
have also been derived in cell culture and therefore new classes
of potential inhibitors can be evaluated against drug-resistant
HIV strains [11].
Difficulties associated with drug-delivery have been observed
mostly in peptide-based drugs due to their highly hydrophilic nat-
ure which results in poor membrane permeability [3,4,12]. A sec-
ond limitation of peptide drugs is their susceptibility to in vivo
degradation.
Over the past few years it was reported that the incorporation
of polycyclic cage frameworks into biologically active molecules
often improves the activity due to enhanced transport across cellu-
lar membranes [13–17]. The lipophilic nature of the cage skeleton
also enables the molecules to cross the blood–brain barrier as well
as the central nervous system (CNS) [14,18–20]. A second advan-
tage of incorporating the cage skeleton into such compounds is
an increased resistance to biodegradability [13]. The computa-
tional design [21–34], NMR elucidation [35–41] and biological
studies [42–45] of several types of polycyclic cage molecules were
previously reported in our group.
⇑
Corresponding authors.
E-mail addresses: mess20@bath.ac.uk (M.E.S. Soliman), maguireg@ukzn.ac.za
(G.E.M. Maguire).
0045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2011.08.002

20
R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
We have recently reported a family of PCU lactam peptides as
potential HIV–PR inhibitors [33,34]. EASY–ROESY NMR experi-
ments were used to determine crucial information about the 3D
structure of these peptides in solution. The active inhibitors exhib-
ited a turn of the peptide side chain that interacts with the cage
protons. Docking of the cage peptides with the C-SA PR was also
performed and a correlation of the binding energies with the IC50
inhibition results was evident. It was also demonstrated for the
first time that the chirality of the PCU moiety was important for
inhibition activity. Although the inhibitors approached activities
in the nano molar range, it was clear that the need for better cage
derived inhibitors should be explored. We decided to investigate a
different PCU derivative as a potential transition state analog as
well as to assess whether the introduction of peptoids may have
a positive effect on the inhibition activity.
Considerable effort has been invested to improve the pharma-
cological properties of peptides, mainly by increasing their enzy-
matic stability while preserving the chemical moieties required
for functional activity [46,47]. Peptoids represent a class of oligo-
meric compounds that closely mimic the natural structure of pep-
tides and possess increased enzymatic stability as compared with
homologous peptides. Peptoids are composed of N-substituted gly-
cine (NSG) residues. The advances in the synthetic strategies used
to prepare peptoid oligomers make them an attractive class of pep-
tidomimetics [48–51].
H2N
OH
H
H
N
O
N
S
O
O
O
O
Ph
non-scissile conformationally
constrained cyclic ether bond
Amprenavir
1
S3
P3
S1
O
O
P1
H
N
H
N
OH
O
N
H
N
H
O
P2
S2
O
stable peptide
non-scissile
bond
conformationally
constrained cyclic
ether bridge
2
Fig. 1. Structure of the proposed 5-hydroxy-4-oxahexacyclo[5.4.1.02,6.03,10.05,9
.
08,11]dodecane peptide showing structural resemblance to the constrained cyclic
ether bond of Amprenavir.
Herein we report the synthesis and efficacy of peptide and pep-
toid based PCU hydroxy ether derived compounds as potential
HIV-1 protease inhibitors. The strategy used for designing HIV pro-
tease inhibitors is to mimic the transition state analog of the Asp-
induced enzyme cleavage thereby increasing the enzyme’s affinity
for the inhibitor over the peptide substrate.
2. Material and methods
2.1. Experimental
Literature reports indicate that phenylalanine is the most com-
Analytical analysis was performed on an Agilent 1100 HPLC
mon substituent at the S1 subsite and it is found in 40% of Gag-Pol
0
(Waters Xbridge C18 150 mm ꢀ 4.6 mm ꢀ 5
lm) coupled to a UV
detector (215 nm) and an Agilent VL ion trap mass spectrophotom-
gene sequences of the virus [52]. According to literature, the S1/S1
0
subsites accommodate hydrophobic substituents [53]. S2/S2 and
0
eter in the positive mode. Semi-preparative HPLC was carried out on
S3/S3 subsites accommodate less hydrophobic substituents such
a Shimadzu 8A instrument (Ace C18 150 mm ꢀ 21.2 mm ꢀ 5
lm)
as valine and alanine. It is postulated that inhibitors must have
peptidic character with the scissile bond of the substrate being re-
placed by a non-cleavable bond [54,55]. The substrate specificity is
determined by subsites located closely to the scissile bond. Primar-
with a UV/VIS detector (215 nm) and automated fraction collector.
A two-buffer system was employed, utilizing formic acid as the
ion-pairing agent. Buffer A consisted of 0.1% formic acid/H2O (v/v)
and buffer B consisted of 0.1% formic acid/acetonitrile (v/v). High
Resolution Electron Spray Ionization Mass Spectroscopic (HRESIMS)
analysis was performed on a Bruker MicroTOF QII mass spectrome-
ter in positive mode with an internal calibration. Microwave cou-
plings were conducted on a Discovery CEM Liberty microwave
peptide synthesizer. The coupling conditions of the peptide synthe-
sizer were adapted from literature [33,34]. The 13C NMR and 1H
NMR data were recorded on a Bruker AVANCE III 400 MHz spec-
trometer. Some samples were analyzed using a Bruker AVANCE III
600 MHz when higher sensitivity was required. Protease hydrolytic
activity was measured by monitoring the relative decrease in absor-
bance at 300 nm using an Analytik Jena Specord 210 spectropho-
tometer. Optical rotations were measured at room temperature in
dry methanol using a Perkin Elmer Polarimeter-Model 341. All IR
spectra were recorded on a Perkin Elmer Spectrum 100 instrument
with a universal ATR attachment. All the amino acids, resins and
coupling reagents are commercially available and were purchased
from GL Biochem (Shanghai) Ltd. Analytical grade solvents for syn-
thesis were procured from Sigma–Aldrich (South Africa).
0
0
ily the S1/S1 and S2/S2 subsites being responsible for substrate
recognition [56]. Based on this concept, we have designed and syn-
thesized nine novel PCU derived hydroxy acid peptides and pep-
toid analogs. Variations of the amino acid sequence in the
peptide chain were introduced. It was shown that the cage ether
type bond of 2 is virtually non-hydrolysable under conditions that
were harsher than physiological conditions [57].
Ghosh et al. developed nonpeptidyl ligands (Fig. 1) that dis-
played enhanced activity when cyclic ethers were incorporated
into the structures [58–60]. The rationale behind using the cage
ether is that when it is incorporated into a short peptide, this cyclic
hydroxy ether moiety could perhaps serve as a cyclic ether transi-
tion state analog under protease conditions. The structural resem-
blance between the non-scissile cyclic ether and the cage ether is
presented in Fig. 1, (structures 1 and 2). If this proposed interaction
between the cage-peptide and the PR is possible, then it should
result in effective inactivation of the PR [53].
This approach is a supplement to our recent report about the
first family of PCU–lactam peptide HIV–PR inhibitors [33,34]. In
that study, information about the 3D solution structure obtained
from EASY–ROESY NMR techniques of the cage peptide could be
correlated to the inhibitory activity of the peptide. A docking
study followed by a QM/MM/MD study of the inhibitor/HIV–PR
complex, suggested that the hydroxy lactam function of the
PCU interacts with the Asp25/Asp250 residues of the protease
enzyme.
2.1.1. In vitro HIV-1 protease activity
The catalytic activity of the HIV-1 protease [34,61] was moni-
tored following the hydrolysis of the chromogenic peptide
substrate Lys-Ala-Arg-Val-Nle-p-nitro-Phe-Glu-Ala-Nle-NH2. This
substrate mimics the conserved KARVL/AEAM cleavage site

R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
21
between the capsid protein and nucleocapsid (CA-p2) in the Gag
polyprotein precursor .
the dried resin beads (1 mg) and left to stand for 20 min. Gen-
eral washing procedure for manual SPPS: After each step of SPPS
the resin was washed with DCM (3 ꢀ 10 mL), DMF (3 ꢀ 10 mL)
and DCM (3 ꢀ 10 mL).
For this study, the chromogenic substrate was synthesized
using a Discovery CEM Liberty microwave peptide synthesizer on
Rink amide resin. The substrate was cleaved from the resin and
deprotected using 95:5 (v/v) TFA:TIS for 3 h. It was then precipi-
tated using cold ether and purified via reverse phase semi-prepara-
tive HPLC on a Shimadzu instrument and characterized using the
Bruker microTOF-Q II instrument (Table 1 in Supplementary
information).
2.1.4. General procedure for the synthesis of peptides using microwave
power
Stock solutions of amino acids (0.2 mM), DIPEA (1 mM) and
O-benzotriazole-N,N,N0,N0-tetramethyl-uronium-hexafluoro-phos-
phate (HBTU) (2 mM) were prepared and inserted to their appro-
priate reaction vessels on the peptide synthesizer. All peptides
were synthesized on a 0.5 mmol scale using microwave power.
The washing procedure was similar to the one used for manual
SPPS. The standard coupling method for Arginine (Arg) was used
for attachment of the second amino acid to the resin (1 g) preload-
ed with the first amino acid. The standard coupling method was
used for subsequent reactions and for Fmoc deprotection
[33,34,62]. The washing procedures were similar to those used
for manual SPPS.
Final cleavage from the resin was performed manually. The re-
sin bound peptide was washed with DCM (3 ꢀ 10 mL) in a reaction
vessel while nitrogen was bubbled through the solution. A cleavage
mixture of 0.5% (v/v) TFA and 95% (v/v) DCM was added to the
dried resin while nitrogen was bubbled through the solution for
10 min. The resin was washed three times with the cleavage
mixture and the cleaved peptide was removed by filtration and
collected in a flask containing water (100 mL). The filtrate was ex-
tracted several times with DCM so as to remove the peptide from
the water layer. DCM was removed under reduced pressure using
a Teflon pump at 40 °C and affording the peptide as a white
powder.
To determine the concentration of the compounds that resulted
in 50% inhibition (IC50) of HIV-1 protease enzyme activity, the pro-
tein (100 nM) and chromogenic substrate (50
lM) were added into
a 120 L microcuvette containing increasing concentrations of
l
inhibitor in a pH 5.0 buffer (50 mM sodium acetate and 0.1 M
NaCl). Protease hydrolytic activity was measured by monitoring
the relative decrease in absorbance at 300 nm using an Analytik
Jena Specord 210 spectrophotometer. Activity was standardized
using commercially available drugs Atazanavir and Lopinavir. All
cage peptides were soluble in the aqueous buffer solution.
(pH = 5, 50 mM sodium acetate and 0.1 M NaCl).
2.1.2. General procedure for the synthesis of 5-hydroxy-
4-oxahexacyclododecane-3-carboxylic acid, compound 15
Compound 15 was synthesized according to the previously
reported method [57].
2.1.3. General procedure for manual SPPS loading of first amino acid to
2-chlorotrityl chloride resin [33,34]
Activated 2-chlorotrityl chloride resin (1 g, 1.33 mmol) was
swelled in dry DCM (10 mL) for 10 min in a sintered glass reac-
tion vessel. Fmoc-Amino acid-OH (3.99 mmol) in DCM (10 mL)
and N,N-diisopropylethylamine (DIPEA) (6.65 mmol), was added
to the resin and was mixed for 2 h with a stream of nitrogen
bubbles. The solvent was removed by filtration and the resin
was washed with DCM (3 ꢀ 10 mL). A few resin beads (ꢁ5 mg)
were removed from the reaction vessel and dried under vacuum
for an hour. A solution of 20% piperidine in DMF was added to
2.1.5. General procedure for the synthesis of peptoids [48–51]
Activated 2-chlorotrityl chloride resin (1.0 g, 1.33 mmol) was
swelled in dry DCM (10 mL) for 10 min in a sintered glass reac-
tion vessel. Bromoacetic acid (1.6 g, 11.51 mmol) and DIPEA
(2 ml) were added to the resin and the mixture was bubbled
for 2 h via a stream of nitrogen. The solvent was removed by
R1
O
O
ii
Br
i
NH
Cl
O
O
O
3
4
5
iii
R1
N
R1
N
O
O
ii
NH
R2
O
O
Br
O
O
O
7
6
iii
R1
N
O
R1
N
O
R3
N
Br
ii
O
N
O
O
N
N
R1
O
R2
O
9
R2
O
8
iv
R1 = Methyl
R1
R2 = Isopropyl
R3 = Benzyl
O
R3
N
N
R4 = Pcu derived hydroxy acid
HO
N
N
O
R2
O
R4
10
Scheme 1. General synthesis of peptoids: (i) = bromoacetic acid, DIPEA, dry DCM, (ii) = amine, DMF, (iii) = bromoacetic acid, DIC, DMF, and (iv) = 5% TFA, DCM.

22
R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
ii
i
+
HO
COOH
COOH
HO
CN
CN
O
O
HO
O
O
OHHO
14
11
12
13
iii
iv
O
HO
HO
O
O
R
2
R = Peptide/Peptoid
15
Scheme 2. Synthesis of 5-hydroxy-4-oxahexacyclo[5.4.1.02,6.03,10.05,9.08,11]dodecane peptide and peptoid analogs 2: (i) = NaCN, CH3COOH, H2O, 0 °C, (ii) = conc. HCl, reflux,
(iii) = 1,4-dioxane, reflux, and (iv) = HATU, DIPEA, DMF, RT.
filtration and the resin was washed with DCM (3 ꢀ 10 mL) and
DMF (3 ꢀ 10 mL). A solution of the amine in DMF (1 M) was
added to the reaction vessel and bubbled for 1 h with nitrogen.
The solvent was removed by filtration and the resin was washed
with DCM (3 ꢀ 10 mL) and DMF (3 ꢀ 10 mL). Bromoacetic acid
(1.6 g, 11.51 mmol) and N,N0-diisopropylcarbodiimide (DIC)
(2 ml) was then added and the mixture was added to the reac-
tion vessel and bubbled for 2 h using nitrogen. The resin was
washed as described previously followed by the addition of a
solution of the amine (1 M), bromoacetic acid (1.6 g, 11.51 mmol)
in DMF and DIC (2 ml). Final cleavage of the peptoid from the re-
sin was carried out similarly as that of the peptides described
previously.
2.73–2.57 (m, 3H), 2.45 (m, 5H), 1.97–1.95 (m, 1H), 1.80–1.77
(m, 1H),1.44 (d, J = 10.12 Hz, 1H), 0.89–0.84 (m, 6H). 13C NMR
(100 MHz, DMSO, 100 °C): d 171.0, 170.5, 170.3, 137.3, 129.2,
127.9, 126.1, 118.1, 89.5, 79.1, 64.8, 58.2, 57.9, 57.2, 56.1, 53.0,
48.5, 48.1, 46.2, 46.0, 42.7, 42.1, 40.5, 37.3, 30.9, 19.0, 18.0, 15.1,
IR (neat): 3288.5, 2965.7, 1728.2, 1638.4, 1524.0, 1327.6, 1206.3,
1138.62, 949.5, 905.3, 699.0 cmꢃ1; HRESIMS (m/z): Calcd for
C
28H33N3NaO7 ([M+Na]23+) 546.2110. Found 546.2190.
2.1.6.3. Compound 19. ½a D20
ꢂ
= ꢃ33.33 (c 0.21 in MeOD); 1H NMR
(400 MHz, DMSO, 100 °C): d 8.54 (q, J = 6.46, 4.54 Hz, 1H), 8.02 (dd,
J = 9.08, 20.28 Hz, 1H), 7.44 (m, 1H), 7.39–7.28 (m, 5H), 7.26–7.06
(m, 5H), 5.11 (s, 2H), 4.62 (m, 1H), 4.36 (m, 1H), 4.24 (m, 1H);
3.082–2.88 (m, 2H), 2.76–2.53 (m, 3H), 2.53–2.39 (m, 5H) 1.92 (m,
1H), 1.79 (d, J = 10.28 Hz, 1H),1.43 (d, J = 10.16 Hz, 1H), 1.32 (d,
J = 7.32 Hz, 3H), 0.89–0.78 (m, 6H) 13C NMR (100 MHz, DMSO,
100 °C): d 172.2, 170.7, 170.6, 170.5, 170.3, 137.4, 135.9, 129.3,
128.4, 128.0, 127.9, 127.8, 126.2, 118.2, 89.6, 65.9, 58.3, 57.9, 57.0,
56.2, 53.1, 48.5, 48.2, 47.6, 46.3, 46.1, 45.9, 42.1, 41.0, 40.1, 37.4,
31.1, 19.0, 18.1, 16.7, IR (neat): 3287.8, 2964.4, 1714.9, 1640.5,
1524.1, 1327.2, 1205.2, 1138.4, 737.7, 697.0 cmꢃ1; HRESIMS (m/z):
Calcd for C36H41N3NaO7 ([M+Na]23+) 650.2837. Found 650.2828.
2.1.6. General procedure for coupling of peptides and peptoids to
5-hydroxy-4-oxahexacyclododecane-3-carboxylic acid, compounds
16–24
The cleaved peptide (1.2 eq.) was dissolved in DCM (3 mL)
followed by the addition of 5-hydroxy-4-oxahexacyclododecane-
3-carboxylic acid[57] (1 equiv), HATU (2.5 equiv) in DMF (7 mL)
and DIPEA (3 equiv) as a base. The mixture was left to stir at room
temperature for 24 h. The product was evaporated to dryness
under vacuum using a Teflon pump at 40 °C. A cleavage mixture
(10 mL) of 95% (v/v) TFA and 5% (v/v) DCM was added to the cou-
pled peptide and stirred for 24 h at room temperature to remove
the N-Boc protecting group. The TFA was removed from the mix-
ture by bubbling air through the peptide and the remaining DCM
was removed under vacuum at 30 °C. The product was obtained
as yellow oil which was purified by preparative HPLC.
2.1.6.4. Compound 20. ½a D20
ꢂ
= ꢃ20.00 (c 0.20 in MeOD); 1H NMR
(400 MHz, DMSO, 100 °C): d 8.43 (m, 1H), 6.91 (q, J = 9.24,
1.68 Hz, 1H), 4.26–4.15 (m, 2H), 2.75–2.66 (m, 4H), 2.51 (m, 2H),
2.47 (m, 2H), 1.97–1.94 (m, 1H), 1.82 (q, J = 9.98, 3.14 Hz, 1H),
1.46 (d, J = 10.16 Hz, 1H), 1.27–1.24 (m, 3H), 0.87–0.77 (m, 6H).
13C NMR (100 MHz, DMSO, 100 °C): d 173.8, 170.4, 118.1, 89.6,
58.6, 57.9, 56.2, 55.9, 48.8, 48.1, 47.4, 46.3, 42.7, 42.1, 41.0, 40.0,
31.5, 19.0, 17.7, 16.9, 15.1, IR (neat): 3288.6, 2970.5, 1726.1,
1638.0, 1529.2, 1454.7, 1326.9, 1292.2, 1204.7, 1137.1, 992.5,
950.2, 906.4, 868.8, 641.7, 521.5 cmꢃ1; HRESIMS (m/z): Calcd for
2.1.6.1. Compound16. ½a D20
ꢂ
= ꢃ23.81 (c 0.21 in MeOD); 1H NMR
(400 MHz, DMSO, 100 °C): d 8.36 (q, J = 7.16, 5.40 Hz, 1H), 7.99
(dd, J = 9.12, 21.73 Hz, 1H), 7.44 (m, 1H), 7.23–7.09 (m, 5H), 4.59
(m, 1H), 4.24–4.15 (m, 2H), 3.05–2.89 (m, 2H), 2.73–2.56 (m,
3H), 2.45–2.43 (m, 6H) 1.97–1.92 (m, 1H), 1.79–1.76 (m, 1H),1.43
(d, J = 10.16 Hz, 1H), 1.27 (d, J = 7.32 Hz, 3H), 0.89–0.83 (m, 6H).
13C NMR (100 MHz, DMSO, 100 °C): d 170.8, 170.5, 170.4, 137.3,
129.3, 127.8, 126.2, 118.1, 89.5, 58.2, 57.9, 57.0, 56.1, 53.0, 48.4,
48.1, 47.4, 46.2, 46.0, 45.9, 42.7, 42.1, 40.9, 37.3, 31.1, 19.0, 18.1,
16.9, IR (neat): 3277.7, 2966.7, 1721.5, 1637.6, 1528.4, 1454.6,
1350.3, 1226.6, 1141.49, 911.2, 743.8, 698.9 cmꢃ1; HRESIMS (m/
z): Calcd for C29H35N3NaO7 ([M+Na]23+) 560.2367. Found 560.2351.
C
20H26N2NaO6 ([M+Na]23+) 413.1683. Found 413.1687.
2.1.6.5. Compound 22. ½a D20
ꢂ
= ꢃ30.00 (c 0.21 in MeOD); 1H NMR
(400 MHz, DMSO, 100 °C): d 8.14 (t, J = 7.98 Hz, 1H), 8.01 (d,
J = 6.72 Hz, 1H), 7.84 (q, J = 8.90, 3.82, 1H), 7.36 (dd, J = 8.16, 2.32,
1H), 4.39–4.21 (m, 4H), 3.70 (m, 4H), 2.78–2.60 (m, 4H), 2.49–2.47
(m, 2H), 2.18 (q, J = 17.02, 8.25, 2H), 1.92–1.69 (m, 4H), 1.46 (d,
J = 10.16 Hz, 1H), 1.43–1.48 (m, 1H), 1.19–1.16 (m, 3H), 1.10–
1.02(m, 1H), 0.84–0.77 (m, 6H). 13C NMR (100 MHz, DMSO,
100 °C): d 173.9, 171.8, 170.9, 170.8, 170.4, 118.2, 89.6, 61.3, 58.4,
58.2, 56.6, 56.3, 54.6, 51.1, 48.6, 48.4, 48.0, 46.4, 46.1, 42.8, 42.2,
41.1, 37.0, 29.9, 27.7, 24.1, 18.1, 15.2, 11.1. IR (neat): 3281.1,
2967.6, 1717.7, 1633.9, 1525.4, 1452.1, 1330.4, 1205.7, 1138.8,
1056.7, 993.7, 950.4, 904.2, 868.6, 789.4, 640.8, 616.9 cmꢃ1. HRE-
2.1.6.2. Compound 17. ½a D20
ꢂ
= ꢃ27.27 (c 0.22 in MeOD); 1H NMR
(400 MHz, DMSO, 100 °C):
d 8.38 (m,1H), 8.04 (q, J = 9.06,
17.98 Hz, 1H), 7.41 (m, 1H), 7.25–7.10 (m, 5H), 4.62 (m, 1H), 4.24
(m, 1H), 3.84–3.71 (m, 2H), 3.36 (m, 1H), 3.06–2.92 (m, 2H),

R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
23
SIMS (m/z): Calcd for C29H40N4NaO11 ([M+Na]23+) 643.2586. Found
643.2579.
1742.8, 1643.5, 1524.4, 1455.5, 1347.2, 1203.2, 1160.4, 1138.8,
1052.4, 991.3, 949.3, 904.7, 738.0, 697.3, 640.9 cmꢃ1; HRESIMS
(m/z): Calcd for
C ) 503.2153. Found
27H32N2NaO6 ([M+Na]23+
2.1.6.6. Compound 23. 1H NMR (400 MHz, DMSO, 100 °C): d 4.75–
4.69 (m, 1H), 4.45–4.35 (m, 1H), 4.15 (s, 1H), 3.97–3.95 (m, 2H),
3.84 (d, J = 4.32 Hz, 1H), 3.01 (d, J = 16.76 Hz, 2H), 2.96–2.89 (m,
2H), 2.79 (d, J = 9.56 Hz, 1H), 2.68–2.56 (m, 2H), 2.47–2.34 (m,
4H), 1.83(d, J = 10.08 Hz, 1H), 1.45(d, J = 10.52 Hz 1H), 1.05–0.99
(m, 6H). 13C NMR (100 MHz, DMSO, 100 °C): d 170.7, 168.2,
118.6, 90.5, 58.3, 55.2, 49.1, 48.3, 46.7, 45.6, 42.9, 41.0, 41.6,
41.0, 40.8, 37.8, 35.2, 34.5, 20.7, 19.3, 18.7. IR (neat): 3358.2,
2968.3, 2866.4, 1728.1, 1603.1, 1460.6, 1405.6, 1335.5, 1290.9,
1195.7, 1134.4, 1068.2, 1002.7, 904.5, 868.5, 827.9, 741.8, 645.7,
503.2150.
2.2. Computational simulations
2.2.1. Preparation of PCU derived peptide inhibitors
The structures of the cage-peptides/peptoids, 16–24, were con-
structed and the geometry was optimized using the MMFF94 force
field implemented in the Avogadro software [64]. Both diastereo-
mers of the cage peptides were investigated. The minimized
structures were then subjected to docking studies.
538.9 cmꢃ1; HRESIMS (m/z): Calcd for C20H26N2NaO6 ([M+Na]23+
)
413.1683. Found 413.1682.
2.2.2. C-SA PR enzyme model system
Since the X-ray structure of the South African HIV-1 protease
subtype C (C-SA) has not yet been reported, the initial 3D structure
of the enzyme was taken from the reported X-ray data of subtype B
HIV–PR (PDB accession code 1HXW) [10]. C-SA HIV–PR differs from
subtype B at eight positions; T12S, I15V, L19I, M36I, R41K, H69K,
L89M and I93L [61]. The X-ray crystallographic coordinates of
1HXW were modified as follows:
The original substrate included in the crystal structure, Ritona-
vir, and the crystallographic water molecules were removed and
hydrogens were added to the system. Ionization states for ioniz-
able amino acid residues were determined according to their stan-
dard pKa values. Mutations at eight positions, T12S, I15V, L19I,
M36I, R41K, H69K, L89M and I93L were manually induced.
Throughout this work, the modeled C-SA HIV–PR was used for
docking and MD simulations. This constructed enzyme was suc-
cessfully used in our previous study on cage lactam peptides
[33,34].
2.1.6.7. Compound 24. 1H NMR (400 MHz, DMSO, 100 °C): d 7.35–
7.14 (m, 5H), 4.69–4.35 (m, 3H), 4.18–4.05 (m, 3H), 3.99–3.89 (m,
4H), 3.05–2.97 (m, 5H), 2.81–2.72 (m, 1H), 2.66–2.65 (m, 2H), 2.47
(s, 2H), 1.87–1.82 (m, 1H), 1.49–1.45 (m, 1H), 1.05–0.91 (m, 6H).
13C NMR (100 MHz, DMSO, 100 °C): d 170.6, 170.2, 169.0, 137.3,
128.4, 127.4, 118.7, 90.5, 58.7, 55.3, 48.6, 47.9, 45.6, 44.5, 42.9,
42.6, 41.8, 40.9, 35.2, 35.1, 20.33, 19.4, 19.2. IR (neat): 3332.8,
2971.8, 1731.5, 1629.7, 1453.7, 1405.5, 1343.5, 1291.8, 1197.6,
1135.9, 1075.7, 1007.0, 904.9, 868.7, 741.1, 630.7 cmꢃ1; HRESIMS
(m/z): Calcd for
560.2358.
C ) 560.2367. Found
29H35N3NaO7 ([M+Na]23+
2.1.7. General procedure [63] for the synthesis of benzyl protected 5-
hydroxy-4-oxahexacyclododecane peptides, 18 and 21
5-Hydroxy-4-oxahexacyclododecane peptide (1 eq) and ben-
zybromide (2.5 eq) were dissolved in DMF (8 mL). Cesium carbon-
ate (2 eq) was added to the solution at ambient temperature. After
stirring for 2 h, saturated aqueous sodium bicarbonate (100 mL)
was added to the solution and extracted with ethyl acetate
(3 ꢀ 100 mL). The combined organic phases were washed with
5% aqueous citrate and saturated aqueous sodium chloride
(100 mL) and dried over sodium sulfate. The solvent was evapo-
rated, and the crude peptide was precipitated in cold ether and
dried under reduced pressure. This crude product was further puri-
fied by preparative HPLC.
2.2.3. Docking of the inhibitors into the C-SA PR model
The energy minimized inhibitors, 16–24 were used in docking
simulations. The effect of ionization states of docked compounds
to the binding scores has been discussed in the literature [65–
67]. The ionization states of the cage-peptide inhibitors have not
yet been determined experimentally. During docking, the neutral
and ionized states (aliphatic amine and carboxylic acid groups of
compounds to be docked were protonated and deprotonated,
respectively) were considered and compared, separately. The sim-
ulations were performed under physiological pH conditions, which
require the correct protonation state of ionizable groups. In partic-
ular, one of the aspartates (Asp25) of the catalytic site exhibits an
increased pKa value of 5.2 in the inhibitor-bound protease [53]
while no increased pKa was reported for the free form of the pro-
tease (pKa = 4.5) [68]. Therefore, a monoprotonated active site
should be prevalent at physiological pH, and thus this protonation
state was used throughout these docking studies. However, to en-
sure that the results of the simulations do not critically depend on
the protonation state, a control simulation of a cage-bound CSA-
HIV-1 protease with a deprotonated carboxylate oxygen of the
Asp25/Asp250 residues was also performed. We have previously
reported that no significant difference was observed for docking
of the cage lactam peptides with the different protonation states
of the enzyme [33,34].
2.1.7.1. Compound 18. ½a D20
ꢂ
= ꢃ19.05 (c 0.21 in MeOD); 1H NMR
(400 MHz, DMSO, 100 °C): d 8.32 (d, J = 7.36 Hz, 1H), 7.95 (q,
J = 9.06, 17.34 Hz, 1H), 7.39 (m, 1H), 7.27–7.09 (m, 10H), 4.63 (m,
1H), 4.58 (m, 1H), 4.48–4.43 (m, 1H), 4.28–4.23(m, 1H), 3.55 (s,
1H), 3.36 (m, 1H), 3.09–2.89 (m, 4H), 2.71–2.55 (m, 3H), 2.45 (m,
4H), 1.97–1.93 (m, 1H), 1.78 (d, J = 10.32 Hz, 1H),1.43 (d,
J = 10.12 Hz, 1H), 0.85–0.80 (m, 6H). 13C NMR (100 MHz, DMSO,
100 °C): d 172.7, 170.7, 170.4, 170.2, 137.4, 137.3, 129.2, 128.9,
128.1, 127.8, 126.3, 118.1, 89.5, 64.8, 58.2, 57.9, 56.9, 56.1, 53.3,
52.9, 48.5, 48.1, 46.2, 45.9, 42.7, 42.1, 40.9, 37.4, 37.3, 36.5, 31.1,
19.0, 17.8, 15.1, IR (neat): 3289.0, 2965.3, 1720.5, 1641.6, 1523.6,
1326.3, 1206.3, 1138.5, 906.4, 698.1 cmꢃ1; HRESIMS (m/z): Calcd
for C35H39N3NaO7 ([M+Na]23+) 636.2719. Found 636.2680.
2.1.7.2. Compound 21. ½a D20
ꢂ
= ꢃ33.33 (c 0.21 in MeOD); 1H NMR
(400 MHz, DMSO, 100 °C): d 8.59 (t, J = 6.82 Hz, 1H), 7.38–7.30
(m, 5H), 7.13 (d, J = 1.40 Hz, 1H), 6.88 (q, J = 9.26, 3.78 Hz, 1H),
5.10 (s, 1H), 4.36–4.31 (m, 1H), 4.27–4.22 (m, 1H), 2.77–2.63 (m,
4H), 2.56–2.51 (m, 2H), 2.47 (m, 1H), 1.96–1.90 (m, 1H), 1.82 (q,
J = 10.20, 5.60 Hz, 1H), 1.47 (d, J = 9.96 Hz, 1H), 1.31–1.29 (m,
3H), 0.85–0.81 (m, 3H), 0.77–0.73 (m, 3H). 13C NMR (100 MHz,
DMSO, 100 °C): d 172.1, 170.4, 170.3, 135.8, 128.3, 128.0, 127.7,
118.1, 89.6, 65.9, 58.6, 57.9, 56.2,55.9, 55.8, 48.8, 48.1, 47.5, 46.3,
46.0, 42.7, 42.1, 31.5, 19.0, 17.6, 16.7. IR (neat): 3286.7, 2966.8,
Docking studies were performed using the Autodock software
[69]. Geisteger charges were computed and the Autodock atom
types were defined using the Autodock Tools graphical user inter-
face supplied by MGL Tools [70]. The Lamarckian Genetic Algo-
rithm (LGA), which is considered one of the best docking
methods available in Autodock [69,71] was employed. This algo-
rithm yields superior docking performance compared to simulated
annealing or the simple genetic algorithm and the other search
algorithms available in Autodock. The docked conformations of

24
R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
O
N
O
N
O
R
OH
OH
OH
O
O
O
R1
R1
O
O
R=Peptide
16-22
N
N
R2
R3
N
R2
O
O
OH
R1 = Isopropyl
R2 = Methyl
O
23
OH
R1 = Benzyl
R2 = Isopropyl
R3 = Methyl
24
Fig. 2. PCU–peptide and PCU–peptoid based inhibitors reported in this work.
Hs
R'
Ha
Hs
Ha
O
O
4
H
N
3
5
2
6
7
H2N
N
H
10
11
9
8
O
R
O
O
1
R'
HO
OH
O
O
Peptide
R'
R
(a)
(b)
R'
N
N
H
N
R = Peptide / Peptoid
O
R'
Peptoid
R' = Methyl, Isopropyl, Benzyl
Fig. 3. Stereochemistry of the generic cage diastereomers (a) and (b).
each of the diastereomeric ligands were ranked into clusters based
on the binding energy. The top ranked conformations were visually
analyzed. The suitability of the constructed C-SA PR for docking
experiments was tested with a control experiment in which Riton-
avir, was docked into the active site of the modeled enzyme and
compared to the X-ray crystal structure [54] of the Ritonavir-
protease complex.
the best docked binding energy was used. Partial charges and
the force field parameters for the inhibitor were generated using
the Antechamber program [72] in the Amber10 package [73].
These were described by the general Amber force field (GAFF)
[74]. All hydrogen atoms of the proteins were added using the
Leap module in Amber10. The standard AMBER force field for
bioorganic systems (ff03) [75] was used to describe the HIV PR
enzyme parameters. Counter ions were added to neutralize the
complex. Then, the system was solvated using atomistic TIP3P
2.2.4. MD simulations of the inhibitor–enzyme complex
water [76] in
complex.
a cubic box with 8.0 Å distance around the
MD simulations were performed for the inhibitor–enzyme
complex of the most active compound 20. The diastereomer with
Table 1
Inhibition of wild type C-SA HIV-1 protease by PCU derived peptides (16–22) and peptoids (23–24).
Entry
Sr. no.
Compound (R)
MF
% Yield
CLogP
Binding energya
IC50 (l
m)b
1
2
3
4
5
6
7
8
16
17
18
19
20
21
22
HO-Ala-Val-Phe-
HO-Gly-Val-Phe-
Bn-O-Ala-Val-Phe-
HO-Phe-Val-Phe-
HO-Ala-Val-
Bn-O-Ala-Val-
HO-Ser-Ilu-Ala-Glu-
R1 = isopropyl
R1 = benzyl
C29H35N3O7
42
54
46
38
68
51
62
57
43
0.792
0.483
2.623
2.210
ꢃ0.367
1.462
ꢃ2.310
0.138
1.795
ꢃ7.01
ꢃ7.74
ꢃ5.53
ꢃ5.02
ꢃ8.98
ꢃ6.54
ꢃ4.62
ꢃ7.21
ꢃ6.75
2
1
C
C
C
C
C
C
28H33N3O7
36H41N3O7
35H39N3O7
20H26N2O6
27H32N2O6
29H40N4O11
>60
>60
0.6
60
>60
5
>60
0.025
0.004
23 (Peptoid)
24 (Peptoid)
R2 = methyl
C20H26N2O6
C29H35N3O7
9
10
11
R2 = isopropyl
R3 = methyl
Lopinavir
Atazanavir
a
Calculated with Autodock. The diastereomer with the lowest binding energy is reported here. The 3D structures of these docked complexes are available as Supple-
mentary material.
b
IC50 = 50% inhibition constant. Standard deviations (SD) for all the compounds were 62% of the reported IC50 values.

R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
25
The molecular dynamics package Amber10 [73] was used for
the minimization and equilibration protocols. Cubic periodic
boundary conditions were imposed and the long-range electro-
static interactions were treated with the particle-mesh Ewald
method [77] implemented in Amber10 with a non-bonding cutoff
distance of 10.0 Å. The energy minimization was first conducted
using the steepest descent method in Amber10 for 1000 iterations
switched to conjugate gradient for 2000 steps with a restraint po-
tential of 2 kcal/mol Å2 applied to the solute. Then the total system
was freely minimized for 1000 iterations. For the equilibration and
subsequent production run, the SHAKE algorithm [78] was em-
ployed on all atoms covalently bonded to a hydrogen atom, allow-
ing for an integration time step of 2 fs. Harmonic restraints with
force constants 2.0 kcal/mol Å2 were applied to all solute atoms.
A canonical ensemble (NVT) MD was carried out for 70 ps, during
which the system was gradually annealed from 0 to 300 K using
a Langevin thermostat with a coupling coefficient of 1.0/ps. Subse-
quently, the system was equilibrated at constant volume and tem-
perature (300 K) with a 2 fs time step for 100 ps while maintaining
the force constants on the restrained solute. With no restraints im-
posed, a production run was performed for 2 ns in isothermal iso-
baric (NPT) ensemble using a Berendsen barostat [79] with a target
pressure of 1 bar and a pressure coupling constant of 2 ps. The
coordinate file was saved every 1 ps and the trajectory was ana-
lyzed at every 1 ps using the Ptraj module implemented in
Amber10.
NMR and MS techniques. The purity of the peptides were deter-
mined with HPLC and were >98%.
The catalytic activity of the HIV-1 protease was monitored by
following the hydrolysis of the chromogenic peptide substrate
His-Lys-Ala-Arg-Val-Leu-Phe(p-NO2)-Glu-Ala-Nle-Ser as described
before [61]. The use of chromogenic substrates has been estab-
lished for the determination of the HIV-1 protease inhibitors effi-
cacy. The method is used to measure the protease activity by
recording an decrease in absorbance as a result of the hydrolysis
of a chromogenic substrate by the protease [83]. The inhibitory
activities of the synthesized compounds were tested using an UV
spectrophotometric assay in order to determine the IC50 values
for each compound. Biological results of the synthesized substrates
are presented in Table 1.
As we have previously reported [34], it appears in general that
the cage peptides exhibit very little cytotoxicity towards human
cells.
According to previous literature reports, phenylalanine is the
preferred substituent (P1) at the S1 subsite and it is found in 40%
of Gag-Pol gene sequences of the virus [52]. Thus, we decided to
synthesize sequences, with phenylalanine as the first amino acid
0
(P1 substituent), attached to the PCU cage. Even though the S2/S2
0
and S3/S3 pockets are also of a hydrophobic nature, both hydro-
philic and hydrophobic side chains from the Gag and Gag-Pol poly-
proteins can occupy these sites. Based on literature and the HIV
substrate sequence, we envisaged that either valine or alanine
would be the best starting point in the peptide chain for binding
0
0
at the S2/S2 and S3/S3 subsites [33,34].
3. Results and discussion
It was observed that compound 16 (entry 1) showed a moderate
inhibition of 2 lM (IC50). For compound 17 (entry 2), the activity
3.1. Synthesis, spectral characterization and biological activity
increased by onefold, where alanine was replaced by glycine at
0
the proposed P3/P3 position. When more hydrophobic bulky
0
The cage peptides and peptoids were synthesized as described
in the experimental section. In most cases, the peptides/peptoids
were of sufficient purity to be coupled to the PCU derived hydroxy
acid after lyophilization. The peptoid oligomers were synthesized
via solid phase synthesis on activated 2-chlorotrityl chloride resin
3 by attaching bromoacetic acid in the presence of DIPEA in dry
dichloromethane (DCM) affording compound 4. Upon filtration,
the amine solution in DMF was added and mixed under a stream
of nitrogen bubbles to give compound 5. The chain was elongated
by coupling bromoacetic acid in the presence of N,N0-diisopropyl-
carbodiimide (DIC). Similarly the corresponding amines were cou-
pled to the extended chain followed by bromoacetic acid to give
compounds 6–10. The completed peptoids were cleaved from the
resin using 5% trifluoroacetic acid (TFA) in DCM as illustrated in
Scheme 1.
The cage hydroxy acid 15 [57] was synthesized from pentacy-
cloundecane-8,11-dione 11 [80–82] which can be easily obtained
from the photocyclization of the Diels–Alder adduct of cyclopenta-
diene and p-benzoquinone. A solution of the dione 11 in a mixture
of water and acetic acid stirred in an ice bath is treated with an
aqueous solution of sodium cyanide to form a mixture of the cya-
nohydrin (12 and 13) [57]. This cyanohydrin mixture when treated
with concentrated hydrochloric acid gives the tri-hydroxy carbox-
ylic acid 14 [57]. It should be noted that this cage acid is synthes-
ised as a racemate. The resulting cage peptides are therefore a
mixture of two diastereomers, Fig. 3a and b.
groups were incorporated as the P3/P3 substituent, little or no inhi-
bition activity was observed (compound 18, entry 3 and 19, entry
4). The reduced potency of these compounds could be as a result of
0
the bulky hydrophobic residues occupying the S3/S3 subsite. Since
0
0
the S1/S1 and S3/S3 positions are located adjacent to one another in
the cis configuration, incorporation of bulky substituents in both
the subsites could alter the conformational arrangements which
consequently reduces their binding affinity [6,53,56]. This possibil-
ity will be discussed with the presentation of the docking results.
To investigate if the PCU-cage is competing with phenylalanine
for the S1 subsite, phenylalanine was removed from peptide 16
(entry 1), to give compound 20 (entry 5). This resulted in a higher
binding affinity and preference for the active site with an inhibi-
tory activity of 0.6 lM (IC50). This is a 2–4-fold greater activity than
compounds 16 (entry 1) and 17 (entry 2), which suggests that PCU
cage is indeed occupying the S1 subsite. To examine the role of a
hydrophobic group at S3 subsite we synthesized compound 21 (en-
try 6) containing a benzyl protecting group on the C terminal of the
peptide sequence. This reduced binding affinity to the active site
and had no inhibitory affect. This result is similar to that of com-
pound 18 (entry 3).
Compounds 22 (entry 7) containing PCU cage with the natural
HIV protease substrate (PCU-EAIS) showed inhibition activity
greater than 60 lM (IC50). Oligomers of N-substituted glycines
are known to be resistant to proteolysis [48–51]. Keeping this in
mind compounds 23 (entry 8) and 24 (entry 9) were synthesized
to mimic the peptide sequence of compound 20 (entry 5) and 16
(entry 1) respectively. Compound 23 (entry 8) showed inhibition
Tri-hydroxy carboxylic acid 14 was refluxed in 1,4-dioxane to
give the desired hydroxy acid 15. The peptides and peptoids ob-
tained were coupled to 15 with HATU, DIPEA in DMF at ambient
temperature as shown in Scheme 2.
activity of 5
lM (IC50), which is approximately 10-fold less active,
when compared to 20 (IC50, 0.6
lM). No inhibition activity was ob-
The products 16–24 (Fig. 2) were isolated and purified as de-
scribed in the experimental section. It was not possible to separate
the diastereomeric mixtures by preparative HPLC. The correctness
of the cage peptides and peptoids were confirmed with standard
served for compound 24 (entry 9) as compared to compound 16
(entry 1).
From the compounds in Table 1 the best inhibition activity was
obtained for compound 20 (entry 5). The activity is promising

26
R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
Fig. 4. (a) Lowest energy docked structure for compound 20 with C-SA HIV–PR. (b) A closer view showing the binding mode of compound 20 inside the C-SA HIV–PR active
site. Some atoms are deleted for clarity. The two PR monomers are colored as the yellow and green ribbon. The inhibitor and the two Asp25 residues presented as colored
sticks. The 3D presentations for computational results are available as PDB files with the Supporting information.
when compared with Lopinavir (entry 10) but almost two orders of
magnitude less active than Atazanavir (entry 11). To further vali-
date these experimental results, docking studies and MD simula-
tions were performed on the most active compound.
scale. Docking studies are used at different stages in drug discov-
ery such as in the prediction of ligand–receptor complex struc-
tures and also to rank the ligand molecules based upon the
binding energies of the corresponding ligand–enzyme complexes.
Docking protocols aid in elucidation of the most energetically
favorable binding mode of the ligand to the receptor. The
objective of our docking study was to elucidate the potential
interaction mode of the PCU–peptide derivatives with C-SA HIV
PR and to see if a correlation between the binding energies
and the observed IC50 results exists.
Docking of Ritonavir (co-crystallized inhibitor) with subtype B
HIV–PR (PDB accession code 1HXW) [10] was previously per-
formed [33,34] to evaluate the efficacy of Autodock for its use in
docking experiments with this subtype as a target. Acceptable re-
sults were obtained. The interacting amino acids in the C-SA start-
ing structure were conserved in the conformation of the enzyme
3.2. Computational simulations
3.2.1. Docking and MD simulations
To obtain a better understanding of the molecular and biolog-
ical behavior of the synthesized PCU–peptide inhibitors, we em-
barked on a computational investigation involving docking and
MD simulations. First, the PCU–peptides were subjected to dock-
ing studies to explore their binding pattern; second MD simula-
tions of the docked inhibitor enzyme complex were performed
to provide a molecular view of the dynamic behavior of such
inhibitors inside the enzyme active site over a suitable time
Fig. 5. Electrostatic and hydrogen bonding interaction between 20 and the nearby residues of C-SA HIV–PR active site. This plot was created with the Ligplot software [85].

R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
27
Table 2
Selected automated docking results for the synthesized PCU derived hydroxy ether
peptide and peptoid inhibitors and comparison to the experimental HIV–PR inhibition
results. (All of the docked inhibitor–enzyme complex results are available in PDB
format and are provided with the Supplementary material.)
(a)
Compounda
Binding energyb (kcal/mol)
IC50
2
(lM)
16-ac
16-bc
17-ac
17-bc
20-ac
20-bc
21-ac
21-bc
23-ad
23-bd
ꢃ6.95
ꢃ7.01
ꢃ7.10
ꢃ7.74
ꢃ8.98
ꢃ8.01
ꢃ6.54
ꢃ5.92
ꢃ7.21
ꢃ6.61
1
0.6
60
5
a
The stereochemistry for the cage peptide is 8-(R)-11-(R)-PCU-peptide for the
(b)
‘‘a’’ diastereomer and 8-(S)-11-(S)-PCU-peptide for the ‘‘b’’ diastereomer.
b
Energy calculated from Autodock. The 3D structures of the docked complexes
are available as Supplementary material.
c
These two diastereomers were not separated and the IC50 values are for the
mixture.
d
These two enantiomers were not separated and the IC50 values are for the
mixture.
after docking and the variation in orientation of these amino acids
in the binding cavity was unnoticeable. Diastereomers of PCU pep-
tides and peptoids were obtained from the coupling of the enantio-
pure short peptide or peptoids to the racemic PCU hydroxyl acid
[33,34,57,84], Fig. 3a and b.
Docking routines of the new PCU–peptide inhibitors with the
C-SA enzyme were performed. The binding energies of the docked
diastereomeric PCU–peptide inhibitors have been tabulated
(Table 2). The docking results are in reasonable agreement with
the measured IC50 values.
In our previous reports [33,34] it was demonstrated that the
chirality of the PCU cage is important for enhanced inhibition.
The docking results in this study again confirm this observation
[33,34]. This suggests that the diastereomers should ideally be
separated when more promising cage peptide inhibitors are
discovered.
(c)
Peptide Chain
HD2
O3
H3
O4
CO2
O
O
O
ASP25'
ASP25
Docking studies have clearly showed that conserved hydrogen
bonds formed between the cage hydroxy ether moiety and
Asp25/Asp250 of the dimeric catalytic triad residues, Asp25-
Thr26-Gly27 (A/B chains) (Figs. 4 and 5 and the Supplementary
Fig. 6. (a) The potential energy of 20 C-SA HIV–PR complex observed in MD
simulation as function of time. (b) Selected distances obtained from MD
simulations: (1) cage(O4)–Asp25(HD2), (2) cage(O3)–Asp25(HD2) and (3)
cage(H3)–Asp250(OD2). (c) Labeling of some atoms involved in hydrogen bond
formation.
a
information). Such hydrogen bonds anchor the cage skeleton to
0
the S1/S1 subsite causing the adjacent amino acids to shift to
0
0
the S2/S2 and S3/S3 . This could explain the finding that less hydro-
phobic amino acids, such as valine, directly attached to the cage
exhibited higher activity.
complex is preserved and very little movement of the PCU cage hy-
droxy ether inside the enzyme pocket is observed during the
course of the MD run. However, slight changes in the orientation
in the cage inhibitor peptide side chain were observed. As evident
from the analysis of the hydrogen bond interactions along the MD
trajectories, the inhibitor is forming hydrogen bonds between the
cage hydroxy ether moiety and at least one of the two Asp25 car-
boxyl groups (Fig. 6c).
Even though docking calculations cannot provide insight on the
dynamic inhibitor–enzyme interactions, they provided us with a
general picture of the most energetically favorable binding orienta-
tion of inhibitors to the enzyme. To obtain further insight into the
dynamic changes of the docked inhibitors within the enzyme ac-
tive site pocket over time, the lowest energy docked complex of
the inhibitor 20 was subjected to unconstrained MD simulations
(2 ns).
The MD simulations clearly showed that the inhibitor easily fits
into the active enzyme pocket. To assess the quality of our MD sim-
ulations, energetic and structural properties are monitored along
the entire 2 ns MD trajectory of the complex. Fig. 6a presents the
plot of the potential energy of the system as a function of time.
The fluctuation of potential energy is less than 1000 kcal molꢃ1
over the course of the reaction.
The 2 ns averaged backbone RMSDs for the 20 enzyme complex
is 1.16 Å and this is an indication that the generated MD trajectory
of the complex is quite stable. The overall orientation of the
4. Conclusion
Pentacycloundecane derived hydroxy acid peptides have
emerged as a new class of irreversible non-scissile ether bridge
type isosters. The results demonstrate the possibility of employing
the cage peptides as potential protease inhibitors. Docking and MD
0
simulations suggest that the cage or part thereof occupies the S1/S1
subsite. The incorporation of bulky substituents such as phenylal-
0
0
anine at the S2/S2 and S3/S3 subsites can alter the conformational

28
R. Karpoormath et al. / Bioorganic Chemistry 40 (2012) 19–29
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arrangement of the inhibitor which consequently reduces their
binding affinity.
Further optimizations as well as in-depth structural and biolog-
ical studies of the selected PCU derived-peptide/peptoid based pro-
tease inhibitors are the subject of ongoing investigations.
Acknowledgments
[33] M.M. Makatini, K. Petzold, S.N. Sriharsha, M.E.S. Soliman, B. Honarparvar, P.I.
Arvidsson, Y. Sayed, P. Govender, G.E.M. Maguire, H.G. Kruger, T. Govender,
Bioorganic & Medicinal Chemistry Letters 21 (2011) 2274–2277.
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Honarparvar, R. Parboosing, A. Naidoo, P.I. Arvidsson, Y. Sayed, P. Govender,
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(2008) 719–726.
This research was supported by NRF (SA) TG (GUN: 66319), KP
(GUN: 69728), HGK and PIA (SA-Sweden bilateral grant), Aspen
Pharmacare and University of KwaZulu-Natal for financial support.
Y.S would like to thank the Carnegie Corporation of New York and
the NRF for financial support. The authors thank Prof. Jürgen
Schleucher (Umeå University, Sweden) and Mr. Dilip Jagjivan
(UKZN, South Africa) for their assistance with the NMR
experiments.
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bioorg.2011.08.002.
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