Accepted Manuscript

Probing of CD4 binding pocket of HIV-1 gp120 glycoprotein using unnaturalphenylalanine analogues

Xiaobo Yu, Poulami Talukder, Chandrabali Bhattacharya, Nour Eddine Fahmi,Jamie A. Lines, Larisa M. Dedkova, Joshua LaBaer, Sidney M. Hecht, ShengxiChen

PII: S0960-894X(14)01118-4DOI: http://dx.doi.org/10.1016/j.bmcl.2014.10.058Reference: BMCL 22113

To appear in: Bioorganic & Medicinal Chemistry Letters

Received Date: 29 September 2014Revised Date: 13 October 2014Accepted Date: 17 October 2014

Please cite this article as: Yu, X., Talukder, P., Bhattacharya, C., Fahmi, N.E., Lines, J.A., Dedkova, L.M., LaBaer,J., Hecht, S.M., Chen, S., Probing of CD4 binding pocket of HIV-1 gp120 glycoprotein using unnaturalphenylalanine analogues, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.10.058

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Probing of CD4 binding pocket of HIV-1 gp120 glycoprotein using unnatural

phenylalanine analogues

Xiaobo Yu b

, Poulami Talukder

a, Chandrabali Bhattacharya

a, Nour Eddine Fahmi

a, Jamie A.

Lines a, Larisa M. Dedkova

a, Joshua LaBaer

b, Sidney M. Hecht

a, Shengxi Chen

a,*

a Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry,

Arizona State University, 1001 South McAllister Avenue, Tempe, Arizona 85287, USA

b Center for Personalized Diagnostics, Biodesign Institute, and Department of Chemistry and

Biochemistry, Arizona State University, 1001 South McAllister Avenue, Tempe, Arizona 85287,

USA

Corresponding author. Tel.: +1 (480) 727 0388; Fax: +1 (480) 965 0038.

E-mail address: shengxi.chen.1@asu.edu (S. Chen)

Keywords: HIV-1 gp120, Phe43 cavity, phenylalanine derivatives, CD4, microarray

ABSTRACT

CD4-gp120 interaction is the first step for HIV-1 entry into host cells. A highly conserved pocket

in gp120 protein is an attractive target for developing gp120 inhibitors or novel HIV detection

tools. Here we incorporate seven phenylalanine derivatives having different sizes and steric

conformations into position 43 of domain 1 of CD4 (mD1.2) to explore the architecture of the

‘Phe43 cavity’ of HIV-1 gp120. The results show that the conserved hydrophobic pocket in

gp120 tolerates a hydrophobic side chain of residue 43 of CD protein, which is 12.2 Å in length

and 8.0 Å in width. This result provides useful information for developing novel gp120 inhibitors

or new HIV detection tools.

Human immunodeficiency virus (HIV) entry into cells is mediated by the trimeric spikes of

glycoproteins gp120 and gp41 on the surface of the virion.1,2

These spikes are anchored in the

HIV membrane by the transmembrane envelope glycoprotein gp41. The surface of the spike is

the envelope glycoprotein gp120, which has non-covalent interactions with gp41 glycoprotein in

each subunit of the trimeric complex.3

The entry of HIV into host cells begins with the binding of

gp120 envelope protein to cellular CD4 receptors, which is primarily expressed on the surface of

macrophages and T cells.4 This CD4 binding induces a reorganization of the trimeric spikes,

resulting in an outward rotation of each gp120 monomer and rearrangement of the gp41 region to

expose the binding site for gp41 chemokine receptors, leading to the second binding between

gp41 and CCR5 or CXCR4.5,6

This glycoprotein complex allows HIV virus to attach and fuse

with target cells and initiate infection.

As the initial binding envelope glycoprotein for HIV entry, gp120 is an attractive target for

developing novel classes of antiretroviral agents or novel detection methods.7-9

Sequence

analysis of HIV-1 gp120 has identified five conserved regions (C1-C5) interspersed with five

glycosylated variable regions (V1-V5).10

Although the surface is extensively glycosylated, the

CD4-binding domain of gp120 remains intact.11

Therefore, this conserved CD4-binding pocket

of HIV-1 gp120 is an attractive target for developing gp120 inhibitors. X-ray structure analysis

of the CD4-gp120 complex has shown direct interatomic contacts between 22 amino acid

residues of CD4 (742 Å2), and 26 amino acid residues distributed over the whole length of gp120

(800 Å2).

11 This large binding surface for CD4-gp120 interaction results in a high specificity and

affinity. Additionally, there is a ‘Phe43 cavity’ in gp120 which is a large conserved hydrophobic

pocket to bind the Phe43 residue of CD4. It plays a vital role for stabilizing interactions between

CD4 and HIV gp120. A previous study reported a 27-mer peptide, CD4M33, which contained a

biphenylalanine at the position corresponding to Phe43 of CD4.12

The single phenyl group

extension of the unnatural peptide CD4M33 into the conserved hydrophobic interfacial pocket of

gp120 enhanced its neutralization capabilities by roughly 10-fold compared to the natural peptide

containing a phenylalanine moiety at the same position. In recent years, several small molecule

inhibitors also have been developed to target the ‘Phe43 cavity’ of gp120.7,8

Thus, it is of interest

to explore the steric tolerance of the ‘Phe43 cavity’, for the future development of novel

antiretroviral agents or detection methods.

Seven phenylalanine derivatives (1 – 7, Fig. 1) have been elaborated. Five analogues (1 – 5) have

substituents in the para and two (6, 7) in the meta position of the phenylalanine ring. This

permits the study of the effect of hydrophobicity and size of amino acid in position 43, on the

stabilization of the CD4-GP120 binding complex. In the CD4-gp120 complex, only domain 1 of

CD4 binds to gp120. To focus on the specific binding, a soluble form of CD4 domain1 (mD 1.2,

12kD) was used for the incorporation of a series of phenylalanine derivatives in this study. In a

previous study, mD 1.2 protein showed greater binding to HIV-1 gp120 and more solubility than

full size CD4 receptor.13

These properties make this small protein an attractive model for our

study. To facilitate comparison of the affinity to HIV-1 gp120 protein, we fused green

fluorescent protein (GFP) to the N-terminal of mD1.2 as a reporter.

The synthesis of the aminoacylated pdCpA analogues of amino acid 1 and 4 – 7 have been

reported previously.14-16

The aminoacylated pdCpA derivative of amino acid 2 was synthesized

as described in the Supporting Information (Scheme S1). The synthesis of the aminoacylated

pdCpA derivatives of amino acid 3 is shown in Scheme 1. Regioselective lithiation (n-

butyllithium, THF, _78°C) of the Schöllkopf auxiliary produced the lithium enolate which

reacted with 1-[4-(bromomethyl)phenyl]pyrrole (8) and afforded adduct 9 with high

diastereoselectivity.17

Acid hydrolysis (2 N HCl) of 9 provided the amino acid methyl ester,

which was protected as the NVOC carbamate to yield 10 in 37% yield. Saponification of 10

afforded the free acid, which was activated as cyanomethyl ester 11 in 78% overall yield.

Treatment of the cyanomethyl ester of 11 with a solution of the tris(tetrabutylammonium) salt of

pdCpA18

in dry DMF gave the corresponding aminoacylated pdCpA 12 in 51% yield.

The individual protected aminoacylated pdCpA was ligated to a suppressor tRNACUA lacking the

3’-terminal CpA moiety.19

The ligation was mediated by T4 RNA ligase. As shown in Scheme 2

and Fig. S1, the ligation afforded full-length aminoacyl-tRNACUA. The N-NVOC group was

removed using a high intensity mercury-xenon light prior to protein synthesis.20

And the N-

pentenoyl group was removed by treatment with aqueous iodine following a previously reported

procedure.21

The activated tRNAs were employed in a cell-free translation system consisting of

an S30 prepared from E. coli, programmed with the GFP-mD1.2 DNA containing a nonsense

codon (TAG) at position 43 of mD1.2.22,23

Since nonsense codon suppression can only be

accomplished in the presence of the added activated suppressor tRNACUA, the aminoacyl-

tRNACUA uniquely decoded the UAG codon, affording GFP-mD1.2 protein with the

phenylalanine derivatives at position 43. As shown in Fig. 2, the expression yields of modified

proteins ranged from 20 to 60% compared to wild-type GFP-mD1.2. The translated proteins

include a hexahistidine fusion peptide at its N-terminal. After purification with Ni-NTA, the

desired GFP-mD1.2 proteins were obtained for study.

To evaluate the effect of these phenylalanine derivatives on CD4-gp120 binding, we employed a

protein array which has the advantages of high sensitivity and high specificity, while utilizing

only a minute amounts of sample.24-26

Purified gp120 proteins were printed on the amino-

modified microscopic slide in four replicates together with bovine serum albumin (BSA) and

rabbit IgG as the negative and positive controls, respectively. To perform the protein-protein

interaction assay, the array was first blocked with cold PPI blocking buffer (1× PBS, 1% Tween

20 and 1% BSA, pH 7.4) for 2 h at 4 oC. Then same amount of the wild-type and modified GFP-

mD1.2 analogues were added to the array surface using a FAST 16-well incubation chamber.

After washing with PPI wash buffer (1× PBS, 5 mM MgCl2, 0.5% Tween 20, 1% BSA and 0.5%

DTT, pH 7.4), the gp120-mD1.2 complexes formed on the array were detected using rabbit anti-

GFP polyclonal antibody and Alexa555 goat anti-rabbit IgG secondary antibody (see

Supplementary data).

As shown in Fig. 3, the size and orientation of the side chain of the phenylalanine derivatives

played a key role in determining CD4-gp120 binding. When the side chain was increased with a

hydrophobic group (iodo-), or an aromatic ring (benzyl- or pyrrolyl-) at the para-position of

phenylalanine, the mutant mD1.2 proteins containing these amino acids (1 – 3) at position 43 had

stronger binding capacity compared to wild-type mD1.2 containing phenylalanine at the same

position. Among these proteins, the protein containing amino acid 2 proved to have the best fit

for the ‘Phe43 cavity’ of HIV gp120. The binding capacity of modified mD1.2 proteins

containing amino acid 2 increased to 156% compared to wild type. When the side chain was

shorter, as in the case of amino acids 1 and 3, the corresponding modified mD1.2 proteins had a

slightly diminished affinity relative to the mD1.2 protein containing amino acid 2. They had

131% and 136% binding capacity compared to wild type, respectively. The nitrogen heterocycle

in amino acid 3 did not have much effect on CD4-gp120 binding, even though the side chain is

somewhat more hydrophilic than amino acid 2. However, the side chain containing three

benzene rings diminished CD4-gp120 interaction. According the X-ray crystallographic structure

of CD4-gp120 (1GC1), the depth of the ‘Phe43 cavity’ of HIV gp120 is as long as the length of

three linear phenyl rings. Thus, amino acid 4 was tolerated at the hydrophobic interfacial pocket

of gp120; the modified mD1.2 containing the amino acid 4 showed 53% binding capacity

compared to wild-type mD1.2. When the first two benzene rings were docked directly into the

gp120 pocket, the third benzene ring at the meta-position was still tolerated. The modified

mD1.2 containing amino acid 5 showed 59% binding capacity relative to wild-type mD1.2.

Interestingly, having two benzene rings at the meta-position of phenylalanine dramatically

reduced the binding ability of mD1.2 to HIV gp120. The modified mD1.2 containing a meta-

connectivity of the phenyl moieties (6) reduced binding capacity to 26% compared to wild type.

However, when the linear three-benzene-ring diverged 60 degree from the original orientation of

phenylalanine (7), the modified mD1.2 lost almost all of its binding capacity to gp120 (3.7%).

The foregoing results indicated that occupancy of the ‘Phe43 cavity’ of HIV gp120 was limited

to the size and orientation of the side chain of the residue 43 of CD4 protein. The pocket

tolerated three linearly linked benzene rings in length (12.2 Å) and accommodated two benzene

rings in width (8.0 Å) for the aromatic side chain.27

In summary, we have incorporated a series of phenylalanine derivatives into the position 43 of

CD4 protein and explored their effect on HIV gp120 binding capacity. The results offer new

insight into the tolerance of the ‘Phe43 cavity’ of HIV gp120. This hydrophobic pocket tolerated

three benzene rings in length (12.2 Å) and two benzene rings in width (8.0 Å) at residue 43 of

the CD4 protein. This provides useful information for the design of novel gp120 inhibitors or

new gp120 detection agents.

Acknowledgements

The authors thank Dr. Dimiter S. Dimitrov for the mD1.2 plasmid as a gift. The authors also

appreciate the help from Dr. Weizao Chen in the Center of Cancer Research of the National

Cancer Institute during the construction of the new plasmid. This work was performed with the

support of the Bill & Melinda Gates Foundation through the Grant Challenges Explorations

initiative (Grant No. OPP1061337).

Supplementary data

Supplementary data (Full experimental details, 1H,

13C NMR and HRMS data) associated with

this article can be found, in the online version, at http://

References and notes

1. Kowalski, M.; Potz, J.; Basiripour, L.; Dorfman, T.; Goh, W. C.; Terwilliger, E.; Dayton,

A.; Rosen, C.; Haseltine, W.; Sodroski, J. Science 1987, 237, 1351.

2. Lu, M.; Blackow, S.; Kim, P. A. Nat. Struct. Biol. 1995, 2, 1075.

3. Liu, J.; Bartesaghi, A.; Borgnia, M. J.; Sapiro, G.; Subramaniam, S. Nature 2008,

455,109.

4. Wyatt, R.; Sodroski, J. Science 1998, 280, 1884.

5. Wu, L.; Gerard, N. P.; Wyatt, R.; Choe, H.; Parolin, C.; Ruffing, N.; Borsetti, A.;

Cardoso, A. A.; Desjardin, E.; Newman, W.; Gerard, C.; Sodroski, J. Nature 1996, 14,

179.

6. Berger, E. A.; Murphy, P. M.; Farber, J. M. Annu. Rev. Immunol. 1999, 17, 657.

7. Zhao, Q.; Ma, L.; Jiang, S.; Lu, H.; Liu, S.; He, Y.; Strick, N.; Neamati, N.; Debnath, A.

K. Virology 2005, 339, 213.

8. Tintori, C.; Selvaraj, M.; Badia, R.; Clotet, B.; Este, J. A.; Botta, M. ChemMedChem

2013, 8, 475.

9. Lines, J. A.; Yu, Z.; Dedkova, L. M.; Chen, S. Biochem. Biophys. Res. Commun. 2014,

443, 308.

10. Starcich, B. R.; Hahn, B. H.; Shaw, G .M.; McNeely, P. D.; Modrow, S.; Wolf, H.; Parks,

E. S.; Parks, W. P.; Josephs, S. F.; Gallo, R. C.; Wong-Staal, F. Cell 1986, 45, 637.

11. Kwong, P. D.; Wyatt, R.; Robinson, J.; Sweet, R. W.; Sodroski, J.; Hendrickson, W. A.

Nature 1998, 393, 648.

12. Huang, C. C.; Stricher, F.; Martin, L.; Decker, J. M.; Majeed, S.; Barthe, P.; Hendrickson,

W. A.; Robinson, J.; Roumestand, C.; Sodroski, J.; Wyatt, R.; Shaw, G. M.; Vita, C.;

Kwong, P. D. Structure 2005, 13, 755.

13. Chen, W.; Feng, Y.; Gong, R.; Zhu, Z.; Wang, Y.; Zhao, Q.; Dimitrov, D. S. J. Virol.

2011, 85, 9395.

14. Gao, R.; Zhang, Y.; Choudhury, A. K.; Dedkova, L. M.; Hecht, S. M. J. Am. Chem. Soc.,

2005, 127, 3321.

15. Chen, S.; Fahmi, N. E.; Wang, L.; Bhattacharya, C.; Benkovic, S. J.; Hecht, S. M. J. Am.

Chem. Soc. 2013, 135, 12924.

16. Chen, S.; Fahmi, N. E.; Bhattacharya, C.; Wang, L.; Jin, Y.; Benkovic, S. J.; Hecht, S. M.

Biochemistry 2013, 52, 8580.

17. Talukder, P.; Chen, S.; Arce, P. M.; Hecht, S. M. Org. Lett. 2014, 16, 556.

18. Robertson, S. A.; Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G.

Nucleic Acids Res. 1989, 17, 9649.

19. Karginov, V. A.; Mamaev, S. V.; An, H.; Van Cleve, M. D.; Hecht, S. M.; Komatsoulis,

G. A.; Abelson, J. N. J. Am. Chem. Soc. 1997, 119, 8166.

20. Mendel, D.; Cornish, V. W.; Schultz, P. G. Annu. Rev. Biophys. Biomol. Struct. 1995, 24,

435.

21. Lodder, M.; Golovine, S.; Hecht, S. M. J. Org. Chem. 1997, 62, 778.

22. Chen, S.; Zhang, Y.; Hecht, S. M. Biochemistry 2011, 50, 9340.

23. Chen, S.; Wang, L.; Fahmi, N. E.; Benkovic, S. J.; Hecht, S. M. J. Am. Chem. Soc. 2012,

134, 18883.

24. Yu, X.; Schneiderhan-Marra, N.; Joos, T. O. Clin. Chem. 2010, 56, 376.

25. Yu, X.; Bian, X.; Throop, A.; Song, L.; Moral, L. D.; Park, J.; Seiler, C.; Fiacco, M.;

Steel, J.; Hunter, P.; Saul, J.; Wang, J.; Qiu, J.; Pipas, J. M.; LaBaer, J. Theranostics

2014, 4, 808.

26. Angenendt, P.; Glokler, J.; Sobek, J.; Lehrach, H.; Cahill, D. J. J. Chromatogr. A 2003,

1009, 97.

27. The distances were measured in X-ray structure of gp120-CD4-Ab17b complex (PBD

entry 1GC1) using PyMOL software.

Legends to Figures

Figure 1. Structures of phenylalanine derivatives 1 – 7.

Figure 2. Autoradiogram of a 12% SDS-polyacrylamide gel (100 V, 2 h) illustrating the

incorporation of phenylalanine derivatives into position 43 of mD1.2. Lane 1, wild-type GFP-

mD1.2 expression; lane 2, modified GFP-mD1.2 DNA in the presence of abbreviated suppressor

tRNACUA-COH; lane 3, incorporation of amino acid 1; lane 4, incorporation of amino acid 2; lane

5, incorporation of amino acid 3; lane 6, incorporation of amino acid 4; lane 7, incorporation of

amino acid 5; lane 8, incorporation of amino acid 6; lane 9, incorporation of amino acid 7.

Phosphorimager analysis was performed using an Amersham Biosciences Storm 820 equipped

with ImageQuant version 5.2 software from Molecular Dynamics.

Figure 3. Differential binding of mD1.2 analogs to HIV gp120 on protein arrays. (A)

Fluorescent images of mD1.2 analog - gp120 interactions. BSA was used as a negative control

and rabbit IgG was used as a positive control for the detection antibody, respectively. WT: wild-

type GFP-mD1.2; #01 - 07: modified GFP-mD1.2 containing amino acid 1 – 7; NC is the PPI

washing buffer used as the assay control. (B) Comparison of the signal intensity of gp120

interaction with the CD4 of wild type and analogues. MFI: mean of signal intensity. *: p-value ≤

0.01. (C) Differential binding of gp120 protein to CD4 analogs. The relative activities were

calculated using the average value of four replicates of each sample and assuming the wild type

as 100 percent. The details are shown in supplementary Table S1.

Scheme 1. Synthetic route employed for the preparation of pyrrolyl-phenylalanyl-pdCpA 12.

Scheme 2. Strategy employed for the incorporation of pyrrolyl-phenylalanine into mD1.2 at

position 43 and binding with HIV-1 gp120 protein.

Figure 1.

Figure 2.

Figure 3.