Chemically programmed antibodies as HIV-1 attachment Inhibitors

Article abstract of DOI:10.1021/ml400097z

Herein, we describe the design and application of two small-molecule anti-HIV compounds for the creation of chemically programmed antibodies. N-Acyl-β-lactam derivatives of two previously described molecules BMS-378806 and BMS-488043 that inhibit the interaction between HIV-1 gp120 and T-cells were synthesized and used to program the binding activity of aldolase antibody 38C2. Discovery of a successful linkage site to BMS-488043 allowed for the synthesis of chemically programmed antibodies with affinity for HIV-1 gp120 and potent HIV-1 neutralization activity. Derivation of a successful conjugation strategy for this family of HIV-1 entry inhibitors enables its application in chemically programmed antibodies and vaccines and may facilitate the development of novel bispecific antibodies and topical microbicides.

Full text of DOI:10.1021/ml400097z

Letter
pubs.acs.org/acsmedchemlett
Chemically Programmed Antibodies As HIV‑1 Attachment Inhibitors
Shinichi Sato,† Tsubasa Inokuma,† Nobumasa Otsubo, Dennis R. Burton, and Carlos F. Barbas, III*
Department of Molecular Biology and Chemistry and the Skaggs Institute for Chemical Biology and Department of Immunology and
Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: Herein, we describe the design and application
of two small-molecule anti-HIV compounds for the creation of
chemically programmed antibodies. N-Acyl-β-lactam deriva-
tives of two previously described molecules BMS-378806 and
BMS-488043 that inhibit the interaction between HIV-1
gp120 and T-cells were synthesized and used to program the
binding activity of aldolase antibody 38C2. Discovery of a
successful linkage site to BMS-488043 allowed for the synthesis of chemically programmed antibodies with affinity for HIV-1
gp120 and potent HIV-1 neutralization activity. Derivation of a successful conjugation strategy for this family of HIV-1 entry
inhibitors enables its application in chemically programmed antibodies and vaccines and may facilitate the development of novel
bispecific antibodies and topical microbicides.
KEYWORDS: Bioconjugation, anti-HIV agent, chemically programmed antibody, microbicide, entry inhibitor
he retrovirus HIV-1, which causes acquired immune
deficiency syndrome (AIDS), has infected 34 million
mAb could further enhance their activity in vivo through
antibody effector functions such as antibody dependent cellular
cytotoxicity (ADCC) and complement dependent cytotoxicity
(CDC). Recently, we have described the development of
chemically programmed antibodies based on the use of mAb
38C2, an aldolase antibody generated by reactive immunization
by using a 1,3-diketone hapten.22−24 This antibody possesses a
low pKa lysine residue in its binding site that is key to its
aldolase activity that can be site-selectively labeled with N-acyl-
β-lactams to produce a chemically programmed antibody.
Chemically programmed antibodies have duration times after
systemic dosing that depend on the properties of the antibody
rather than on those of the conjugated small molecule,
providing for very significant extensions in the pharmacokinetic
profiles of the attached molecule.18,20 We have demonstrated
the utility of this approach by preparing mAb conjugates that
show promising activity in a variety of cancer models but also in
the area of anti-infectives through the preparation of CCR5
blocking mAbs that inhibit HIV-1 entry and neuraminidase
inhibitors that neutralize influenza.18−20
T
people worldwide, and this number is expected to increase by
2.5 million each year into the near future.1 Although the
combination reverse transcriptase inhibitor/protease inhibitor
treatment known as HAART has proven successful,2,3 side
effects and viral escape are significant issues, and new
treatments are needed. The viral envelope protein gp120, the
primary target for antibody mediated viral neutralization, is an
emerging target for small molecule treatment of HIV
infection.4,5 This protein is responsible for the entry of HIV
into host cells. In the initial step of entry, gp120 binds to the
CD4 glycoprotein expressed on the surface of human immune
cells. Bristol−Myers Squibb Pharmaceutical Research Institute
discovered small molecules BMS-378806 (1) and BMS-488043
(2) that bind to gp120 (Figure 1) and block its interaction with
Treatment as well as prophylaxis of HIV-1 infection requires
the development of a cocktail of inhibitors. In order to
complement our anti-CCR5 blockade based on this strategy,18
we envisioned that the conjugate of mAb 38C2 and the small-
molecule gp120 inhibitor would bind to gp120 and inhibit
CD4-mediated entry of HIV-1 into cells (Scheme 2). In related
work, Spiegel and co-workers recently reported that a derivative
of HIV-1 inhibitor 1 modified with a 1,3-dinitrophenyl hapten
moiety binds to HIV gp120.25 Their compound was designed
to bind noncovalently with polyclonal anti-1,3-dinitrophenyl
Figure 1. Chemical structures of gp120 inhibitors.
CD4.6−11 However, the short pharmacokinetic profiles of these
small molecule inhibitors (half-lives after intravenous injection
are 0.3 and 2.4 h, respectively) may limit their clinical
application.
We hypothesize that the pharmacokinetic properties of these
small molecule gp120 inhibitors can be improved by
conjugation with a monoclonal antibody (mAb) (Scheme
1).12−21 Furthermore, coupling of the small molecule to the
Received: March 8, 2013
Accepted: April 7, 2013
Published: April 8, 2013
© 2013 American Chemical Society
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Scheme 1. Chemoselective Modification of Aldolase Antibody 38C2 to Yield a Chemically Programmed Antibody
Scheme 2. Schematic Representation of the Inhibition of the
HIV Entry by gp120 Inhibitor-Programmed mAb 38C2
presence of CuSO4, tris(3-hydroxypropyltriazolylmethyl)amine
(THPTA), and sodium-(L)-ascorbate proceeded smoothly to
yield desired compound 3 with the linker now at the Northern
sector of the molecule as suggested by Spiegel et al.26
Inhibitor 2 presented us with opportunities to explore the
southern sector of the molecule for attachment. Structure−
activity relationship studies of 29−11 found that bulky
substituents at the 4-position of the azaindole unit decreased
the inhibition activity of the compound. Thus, a northern
sector connection would be ill-advised. Protection at the 1-
position also gave diminished biological activities, whereas the
piperazine of 2 was already optimized. In contrast, substitution
was tolerated at the 7-position of the azaindole. ON the basis of
these data, we designed 4 bearing the linker at 7-position of the
azaindole (southern sector connection).
Target compound 4 was synthesized as shown in Scheme 4.
Commercially available 2-hydroxy pyridine derivative 9 was
subjected to bromination to afford 10 in good yield. The
hydroxy group of 10 was allylated using Ag2CO3. Formation of
the core azaindole structure was achieved by treatment of 11
with N,N-dimethylformamide dimethylacetal followed by
reduction of nitro group in the presence of Fe in AcOH. The
bromo group of 12 was replaced by a methoxy group, and 13
was treated with borane-dimethylsulfide complex followed by
oxidation with hydrogen peroxide to replace the terminal olefin
with a primary alcohol. The reactivity of the substituent-free
nitrogen atom at the 1-position of the azaindole in 14 was
problematic. After analysis of a number of protecting groups,
we found that the trimethylsilylethoxymethyl (SEM) group
could be utilized.27 Protection of the reactive azaindole moiety
yielded 15, which was subjected to etherification with 1628 to
obtain 17. Removal of the SEM group was performed using
tetrabutylammonium fluoride (TBAF). A Friedel−Crafts
reaction of 18 and methyl-2-chloro-2-oxoacetate was accom-
plished in the presence of an excess amount of AlCl3.29 The
resulting compound 19 was hydrolyzed and condensed with 1-
benzoylpiperazine 20 mediated by 3-(diethoxy-phosphory-
loxy)-3H-benzo[d][1.2.3]triazine-4-one (DEPBT)30 to afford
the derivative of BMS-488043 21. As the final step, a Huisgen
reaction was performed under conditions described for
synthesis of 3 to obtain the desired compound 4.
(DNP) antibodies in situ, with the aim of enhancing the activity
of 1. The activity of 1, however, was severely compromised
upon the addition of the DNP linker in their report. Parental 1
has HIV-1 neutralization activity in the nanomolar range,
whereas DNP linked 1 demonstrated micromolar activity in
binding studies and was not shown to neutralize HIV-1. Our
conjugate strategy differs since we use a defined monoclonal
antibody covalently linked to 1. We hypothesized that our
strategy might allow us to recover the potent activity of 1
directly if the lack of activity of their DNP derivative of 1 was
due to the noncovalent nature of attachment to antibody.
Alternatively, modification of the linkage strategy to this family
of inhibitors might be key to restoring the activity of the small
molecule.
To prepare derivatives of the Bristol−Myers Squibb
compounds for conjugation to mAb, we first prepared β-lactam
3 (Figure 2) derived from BMS-378806 (1) from the known
compound 5 (Scheme 3).7 Substitution of the nitro group by
alcohol 6 followed by the treatment of PCl3 gave BMS-378806
derivative 7 bearing an azide group. The Huisgen reaction of 7
with β-lactam 8 possessing a terminal alkyne group in the
Conjugation of agent 3 with mAb 38C2 to form 22a was
carried out by incubating 38C2 with six equivalents of 3 in 10
mM PBS (pH 7.4) at room temperature for two hours (Scheme
5). We evaluated the conjugation by measuring the catalytic
activity of retro-aldol reaction of methodol as per the standard
method.15 Once a conjugate is formed, the antibody cannot
catalyze the retro-aldol reaction of methodol. Compound 22a
had undetectable catalytic activity indicating that each of the
key catalytic lysine residues had reacted with the lactam (Figure
3A). The MALDI-TOF mass analysis of 22a supported the
effective conjugation of 38C2 with 3 (Figure 3B). The
difference in mass between 38C2 and our preparation of 22a
Figure 2. Synthetic targets for this study.
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a
Scheme 3. Synthesis of the BMS-378806 Programming Agent 3
a
Reagents and conditions: (a) NaH, DME, RT, 2 h then 50 °C, 3 h. (b) PCl3, EtOAc, RT, 2.5 h (37% in two steps). (c) CuSO4·5H2O, THPTA, Na-
(L)-ascorbate, tBuOH, H2O, RT, 30 min (57%).
Scheme 4. Synthesis of the BMS-488043 Programming
Agent 21
Supporting Information). Conjugate 22b was similarly
prepared from 4 and 38C2 and characterized (Figure 3A,C).
Initially, the binding of antibody conjugates 22a and 22b to
gp120 was evaluated using an ELISA with gp120-coated plates
(Figure 4). Neither unconjugated mAb or conjugate 22a bound
to gp120 at 200 nM. Signal in these cases was similar to the
negative control of buffer alone (PBS). In contrast, the 22b
bound strongly to gp120 at this concentration as did the
positive control broadly neutralizing antibody b12.31 The lack
of binding by 22b is consistent with the results of the
structure−activity relationship study of related compounds that
the bulky substituent at 4-position of the azaindole 1
diminished the biological activity.9−11 Loss of binding activity
at this concentration is consistent with the reported low
binding activity of the DNP conjugate study and indicates that
the northern site of the linker attachment is likely responsible
for the loss in binding, not the fact that DNP conjugates with
antibodies are reversibly formed.
a
The anti-HIV activities of the conjugates 22a and 22b were
measured in neutralization assays with a single round of
infectious virus (JRFL) as described previously.32 Conjugate
22a showed very weak neutralization activity, consistent with
the low gp120 binding activity observed. Confirming our
hypothesis that the substituent at the northern sector 4-position
of 1 disrupted gp120 binding, neither 3 nor 7 were effective in
the assay (Figure 5A). The IC50 values of 4 and 21 with the
linker at southern 7-position were 67.5 and 25.4 nM,
respectively. The conjugate 22b also blocked infection with
an IC50 of 128 nM (Figure 5B). The unmodified mAb 38C2
had no relevant anti-HIV activity. Evident from these studies is
an impact on activity on linker attachment to the southern 7-
position; however, significant neutralization activity was
preserved following linker addition at this site. We had
anticipated that conjugate 22b might exhibit significantly
enhanced activity over 4 and 21 given the bivalent display of
the compound on the antibody following conjugation as we
have noted with other antibody targeting agents. The lack of
enhanced activity following conjugation suggests that 22b is
unable to engage the HIV-1 virion in a bivalent interaction.
Monovalent binding of natural antibodies that react with the
CD4-binding site on gp120 has been suggested in the
literature.33 As previously reported, the chemically programmed
antibody strategy has been shown to significantly extend the
half-life of the targeting molecule relative to the unconjugated
molecule in studies concerned with small molecule, peptide,
a
Reagents and conditions: (a) Br2, AcOH, AcONa, RT, 1 h (75%). (b)
Ag2CO3, AllylBr, toluene, RT, 16 h (quant). (c) N,N-dimethylforma-
mide dimethylacetal, DMF, 130 °C, 2 h. (d) Fe, AcOH, 100 °C, 90
min (40% in two steps). (e) CuI, MeONa, MeOH, DMF, RT to 110
°C, 19 h (87%). (f) BH3-Me2S, THF, 0 °C to RT, 4 h then H2O2,
NaOH, H2O, 0 °C to RT, 15 h (42%). (g) KOH, SEMCl, THF, RT,
30 min (88%). (h) NaH, DMF, RT, 19 h, (55%). (i) TBAF,
ethylenediamine, THF, RT to 70 °C, 21 h (85%). (j) AlCl3,
ClCOCO2Me, CH3NO2, CH2Cl2, RT, 4 h (40%). (k) NaOH, H2O,
MeOH, RT, 1 h. (l) DEPBT, DIPEA, RT, 10 h (38% in two steps).
(m) CuSO4·5H2O, THPTA, Na-(L)-ascorbate, tBuOH, H2O, RT, 3 h
(69%).
corresponded to two equivalents of the small molecule
derivative of 3. ESI-MS analysis also indicated that both of
the two catalytic lysine moieties of 38C2 were modified (see
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a
Scheme 5. Preparation of the gp120 Inhibitor Programmed Antibodies 22a and 22b
a
Reagents and conditions: (a) PBS (pH 7.4), RT, 2 h.
Figure 3. Analysis of 22a and 22b. (A) Catalytic activity of 22a, 22b, and mAb 38C2 in the retro-aldol reaction of methodol. (B) Overlay of MALDI
mass spectra of mAb 38C2 (blue, MWav = 150 357) and 22a (green, MWav = 152 932). (C) Overlay of MALDI mass spectra of mAb 38C2 (blue,
MWav = 150 357) and 22b (green, MWav = 152 946).
and aptamer targeting molecules.18−21 Additional biological
activities not accessible to the small molecule itself but rather
characteristic of the antibody conjugate would be expected to
be seen in vivo for 22b such as ADCC and CDC activity, and
these activities may be important to the activities of natural
anti-HIV-1 antibodies.34
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discovery of a viable site of conjugation for this promising
family of attachment inhibitors35 has allowed us to establish
good antiviral activity in the case of a chemically programmed
antibody, active conjugation to this family of inhibitors should
also facilitate their application in chemically programmed
vaccines,36 chemical approaches to bispecific antibodies,37 and
topical microbicides whose construction is hereby facilitated.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures, analytical data, and procedures for ELISA
and neutralization assay. This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 4. Binding of mAb 38C2 (200 nM), 22a (200 nM), 22b (200
nM), and mAb b12 (2 nM) to JRFL gp120 as evaluated by ELISA.
PBS indicates the background control.
AUTHOR INFORMATION
Corresponding Author
*(C.F.B.) Tel: 858-784-9098. Fax: 858-784-2583. E-mail:
carlos@scripps.edu.
■
Author Contributions
†These authors contributed equally to this work.
Funding
This work was supported by NIH grant AI095038.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Angelica Cuevas for performing HIV-1 neutralization
assays.
■
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