A mechanism-based probe for gp120-Hydrolyzing antibodies

Article abstract of DOI:10.1016/S0960-894X(02)00640-6

An antigenic peptide analogue consisting of HIV gp120 residues 421-431 (an antigen recognition site probe) with diphenyl amino(4-amidinophenyl)methanephosphonate located at the C-terminus (a catalytic site probe) was synthesized and its trypsin and antibody reactivity characteristics were studied. Antibodies to the peptide determinant recognized the peptidyl phosphonate probe. Trypsin was inhibited equipotently by the peptidyl phosphonate and its simple phosphonate counterpart devoid of the peptide determinant. The peptidyl phosphonate inhibited the gp120-hydrolyzing activity of a catalytic antibody light chain. It was bound covalently by the light chain and the binding was inhibited by the classical active-site directed inhibitor of serine proteinase, diisopropyl fluorophosphate. These results reveal that the peptidyl phosphonate ester can serve as a probe for the antigen recognition and catalytic subsites of proteolytic antibodies.

Full text of DOI:10.1016/S0960-894X(02)00640-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3167–3170
A Mechanism-Based Probe for gp120-Hydrolyzing Antibodies
Hiroaki Taguchi, Gary Burr, Sangeeta Karle, Stephanie Planque, Yong-Xin Zhou,
Sudhir Paul* and Yasuhiro Nishiyama*
Chemical Immunology and Therapeutics Research Center, Department of Pathology and Laboratory Medicine,
University of Texas—Houston Medical School, 6431 Fannin, Houston, TX 77030, USA
Received 31 May 2002; accepted 23 July 2002
Abstract—An antigenic peptide analogue consisting of HIV gp120 residues 421–431 (an antigen recognition site probe) with
diphenyl amino(4-amidinophenyl)methanephosphonate located at the C-terminus (a catalytic site probe) was synthesized and its
trypsin and antibody reactivity characteristics were studied. Antibodies to the peptide determinant recognized the peptidyl phosphonate
probe. Trypsin was inhibited equipotently by the peptidyl phosphonate and its simple phosphonate counterpart devoid of the
peptide determinant. The peptidyl phosphonate inhibited the gp120-hydrolyzing activity of a catalytic antibody light chain. It was
bound covalently by the light chain and the binding was inhibited by the classical active-site directed inhibitor of serine proteinase,
diisopropyl fluorophosphate. These results reveal that the peptidyl phosphonate ester can serve as a probe for the antigen recognition
and catalytic subsites of proteolytic antibodies.
# 2002 Elsevier Science Ltd. All rights reserved.
Site-specific proteolytic Absy hold promise for efficient
removal of microbial protein targets without indis-
criminate hydrolysis of other proteins. Certain proteolytic
Abs contain active sites consisting of a serine proteinase-
like catalytic triad that is functionally coordinated with
traditional noncovalent antigen binding forces, allowing
antigen-specific stabilization of the ground state followed
by nucleophilic attack on the antigenic substrate.1 Several
serine proteinase-like Abs were identified in patients
with immunological disorders and animals immunized
with polypeptide antigens.2a—d Compounds that bind
such Abs selectively are essential for study of catalytic
mechanism and for capture of catalysts from displayed
Ab repertoires. Recently, simple phosphonate diesters
have been developed as probes for nucleophiles present
in displayed Ab and non-Ab serine proteinases,3a,b and
phosphonate monoester transition state analogues have
previously been employed for isolation of certain esterase
Abs.4 The mechanism-based covalently reactive com-
pounds are the preferred probes for serine proteinase-
like Abs, since they recapitulate the covalent reaction of
peptide substrates with the enzyme active site.5 The
ideal mechanism-based probes, however, should contain
a structural component designed to bind the Abs with the
desired antigenic specificity in addition to the chemically
reactive phosphonate. Here we describe the design,
synthesis and Ab reactivity characteristics of a covalently
reactive antigenic peptide analogue (CRA) consisting of
a phosphonate diester moiety (the catalytic subsite
probe) and a peptide derived from the conserved region
of the HIV coat protein gp120 (the Ab recognition site
probe).
yAbbreviations: Ab, antibody; BSA, bovine serum albumin; CRA,
covalently reactive antigenic peptide analogue; DFP, diisopropyl
fluorophosphate; EAR-MCA, N-tert-butoxycarbonyl-g-benzyl-Glu-
Ala-Arg-7-amino-4-methylcoumarin hydrochloride; HIV, human
immunodeficiency virus type 1; IgG, immunoglobulin G; KLH, key-
Design
Diphenyl amino(4-amidinophenyl)methanephosphonate
derivatives are active-site directed irreversible inhibitors
of trypsin-like enzymes,6 forming the covalent enzyme-
inhibitor adducts by phosphonylating the active site Ser
residue.7 The positively charged amidino group adjacent
to the phosphonate diester group serves as an analogue
of Lys432 of gp120. The Lys432–Ala433 bond is the
most likely specific cleavage target by Abs because (1)
hole
lympet
hemocyanin;
PyBOP,
(benzotriazol-1-yl)oxy-
tris(pyrrolidino)phosphonium hexafluorophosphate; SDS, sodium
dodecylsulfate; TFE, 2,2,2-trifluoroethanol. Amino acids, peptides
and their derivatives were abbreviated as recommended by the
IUPAC–IUB Joint Commission on Biochemical Nomenclature: Eur.
J. Biochem. 1984, 138, 9.
*Corresponding authors. Fax:+1-713-0574; e-mail: yasuhiro.nishiyama@
uth.tmc.edu (Y. Nishiyama); sudhir.paul@uth.tmc.edu (S. Paul).
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00640-6

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H. Taguchi et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3167–3170
most natural proteolytic Abs cleave Lys/Arg–X bonds
(X represents an amino acid residue);8a,b (2) this bond is
located in a region responsible for HIV binding by host
CD4 receptors, and it’s hydrolysis by trypsin is shown
to induce considerable loss of the CD4-binding cap-
ability of gp120.9 As to the peptide component, Abs can
recognize peptides as short as 4–5 amino acids, but high
affinity binding is usually attained with somewhat longer
peptides (>10 residues).10 Thus the undecapeptide cor-
responding to gp120 residues 421–431 was linked to an
amino function located in the phosphonate unit (Fig. 1).
The resulting molecule is intended to serve as a message–
address system in which the gp120-derived peptide
(address) delivers the phosphonate moiety (message) to
the catalytic site. Biotin and Cys were introduced to the
N-terminus to permit detection of the molecule and its
conjugation to carrier proteins, respectively.
Synthesis
The common stepwise coupling of protected amino
acids onto the phosphonate derivative was not
employed to avoid potential hydrolytic decomposition
of the terminal phosphonate ester moiety by successive
exposure to harsh deprotection conditions. Instead,
CRAs 1a–c were synthesised by condensing the protected
peptide segments (4a–c) and the aminoalkanephospho-
nate derivative as outlined in Figure 1b. Briefly, inter-
mediate 3 was prepared by Fmoc-based solid-phase
synthesis on 2-chlorotrityl resin followed by cleavage with
30% TFE–CH2Cl2.11 Then biotin tags with cleavable and
non-cleavable linkers and Boc-Cys(Trt) were incorporated
to give 4a–c. These were coupled with diphenyl amino
(4-amidinophenyl)methanephosphonate using PyBOP.
The products were deprotected with anhydrous TFA to
give CRAs 1a–c, which were purified by reversed-phase
HPLC to homogeneity.12 1c was conjugated with BSA
using a commercially available bifunctional cross-linking
agent, N-(g-maleimidobutyryloxy)succinimide. Average
mol/mol ratio of 1c and BSA was 3.9.13 Compound 2 was
synthesized as described in the previous report.14
Ab Reactivity Characteristics
To determine the integrity of the antigenic determinant
in 1a, its binding to Abs raised to a KLH conjugate of
gp120(421–436)15 was measured by ELISA. Various
dilutions (1:100–1:10000) of the antiserum from BALB/c
mice immunized with gp120(421–436)–KLH were incu-
bated in wells containing immobilized 1c–BSA and
gp120(421–436)–BSA, and Ab-binding was measured
using an anti-IgG–horseradish peroxidase conjugate. The
anti-gp120(421–436) Abs were bound by immobilized 1c–
BSA and gp120(421–436)–BSA with near equivalent
potency (Fig. 2a). Importantly, binding of 1c–BSA by
nonimmune IgG was no greater than of gp120(421–
436)–BSA, indicating that the presence of the phosphonate
group does not result in nonspecific Ab phosphonylation.
This result indicates that the antigenic peptide component
of 1c is indistinguishable from the peptide devoid of the
phosphonate group.
Figure 1. (a) Design of CRAs 1a–c. CRAs consist of the peptide
derived from CD4-binding region of gp120 (Ab recognition site probe)
and the chemically reactive phosphonate moiety (catalytic site probe).
Biotin and Cys were introduced to the N-terminus to permit detection
of the molecule and its conjugation to carrier proteins, respectively. (b)
Synthetic scheme for CRAs 1a–c. Reagents and conditions: (i) TFE–
CH2Cl2 (3:7); (ii) (2-biotinamido)ethylamido-3,30-dithiodipropionic
acid N-hydroxysuccinimide ester, iPr2EtN, 1-methyl-2-pyrrolidinone;
(iii) (6-biotinamido)hexanoic acid N-hydroxysuccinimide ester, iPr2EtN,
1-methyl-2-pyrrolidinone; (iv) Boc-Cys(Trt) N-hydroxysuccinimide
ester, iPr2EtN, 1-methyl-2-pyrrolidinone; (v) diphenyl amino(4-amidi-
nophenyl)methanephosphonate dihydrobromide, PyBOP, iPr2EtN,
DMF; (vi) TFA. P represents the solid support.
To assess the reactivity of 1a with the active site
nucleophiles, its interactions with two catalysts were
studied: (1) the serine proteinase trypsin and (2) an Ab
light chain previously identified to display gp120ase

H. Taguchi et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3167–3170
3169
The covalent adduct of 1a with the light chain was
detected as a band at 25 kD by denaturing electrophor-
esis (Fig. 2d). Pretreatment of the light chain with DFP
substantially reduced formation of the 1a-adduct band,
indicating that a serine proteinase-like catalytic site is
responsible for 1a-binding. These observations indicate
uncompromised chemical reactivity of the phosphonate
moiety in 1a.
Conclusion
These results indicate that CRAs 1a–c are suitable
probes for Abs that combine a serine proteinase-like
catalytic mechanism with the ability to recognize a con-
served antigenic determinant of gp120. Such CRAs can
be applied for identifying gp120-hydrolyzing Abs as stable
covalent adducts from autoimmune Ab repertoires, which
are known to contain gp120 specificities.17 Furthermore,
CRA–protein conjugates such as 1c–BSA reported here
are potential immunogens to induce synthesis of proteo-
lytic Abs specific for gp120.
Figure 2. (a) 1c–BSA binding by anti-gp120(421–436)–KLH Ab.
Maxisorp 96-well microtiter plates were coated with BSA–gp120(421–
436) and 1c–BSA (11.2 pmol peptide equivalents/well) in 100 mM
bicarbonate buffer (pH 8.6, 2 h, 37 ꢀC). Wells were washed three times
at each step with 10 mM sodium phosphate containing 137 mM NaCl,
2.7 mM KCl and 0.05% Tween-20 (pH 7.4, PBS-T) and blocked with
5% skim milk in PBS-T. Anti-gp120(421–436)–KLH serum obtained
from a mouse and nonimmune mouse serum were diluted in PBS-T
containing 1% skim milk (binding buffer, 1:100, 1:300, 1:1000, 1:3000,
1:10,000). Bound murine IgG was detected using peroxidase-con-
jugated goat anti-mouse IgG diluted in binding buffer. Histogram
shown is data using 1:1000 dilution sera. (b) Inhibition of trypsin by
1a. Trypsin (0.75 nM) was preincubated in 10 mM sodium phos-
phate, pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, and 0.025%
Tween-20 in the presence of 1a or 2 (0.25 mM–4.0 mM, 37 ꢀC, 30
min). EAR-MCA was added to the solutions (final concentrations:
trypsin, 0.6 nM; inhibitor, 0.1 mM–0.4 mM; substrate, 0.2 mM), and
amidolytic activity was determined by fluorimetric measurement of
7-amino-4-methylcoumarin generated at 470 nm with excitation at
360 nm. % Inhibition was calculated relative to fluorescence without
inhibitor (616 FU/h). Background fluorescence in the absence of
enzyme was 31 FU/h. (c) Inhibition of Ab light chain catalyzed 125I-
gp120 cleavage by 1a. Inset, autoradiogram showing non-reducing
SDS-electrophoresis gels of Ab light chain (LAY, 0.5 mM) catalyzed
hydrolysis of 125I-gp120 (1.1 nM, 2.3ꢁ103 cpm) in the presence of 1a
(10 mM; lane 3) or 2 (10 mM; lane 4). Lanes 1 and 2 show 125I-gp120
incubated in assay diluent with and without the light chain (0.5 mM),
respectively. 17 h, 37 ꢀC; pretreated with inhibitor for 1 h. Plot shows
inhibition of the cleavage reaction at varying 1a concentrations calcu-
lated as the decrement of 80 kD-band intensity determined by densi-
tometry. (d) Covalent 1a binding by Ab light chain. Streptavidin-
peroxidase stained blots of SDS-gels showing reaction mixtures of 1a
(80 mM) treated for 1 h at 37 ꢀC with light chain LAY (1.0 mM) with-
out (lane 1) and with DFP pretreatment (lane 2; 5.0 mM, 37 ꢀC, 0.5 h).
Acknowledgements
This work was supported by United States Public
Health Service Grants AI31268 and AI46029, and Naito
Foundation, Japan (YN).
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13. 1c/BSA ratio was calculated from reacted SH (determined