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. 2000 May 9;97(10):5639-44.
doi: 10.1073/pnas.090576697.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

T Dragic  1 , A Trkola, D A Thompson, E G Cormier, F A Kajumo, E Maxwell, S W Lin, W Ying, S O Smith, T P Sakmar, J P Moore
Affiliations

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

T Dragic et al. Proc Natl Acad Sci U S A. .

Abstract

HIV-1 entry into CD4(+) cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

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Figures

Figure 1
Figure 1
Effect of TAK-779 on HIV-1 replication and Env-mediated membrane fusion. (A) Replication of HIV-1JR-FL in mitogen-activated peripheral blood mononuclear cells was measured in the presence of TAK-779 (■). (B) Fusion between CHO-K1-CD4-CCR5 cells and HeLa cells expressing the HIV-1JR-FL Env was measured in the presence of TAK-779 (■) or RANTES (□). The extent of inhibition of viral replication or cell–cell fusion at each inhibitor concentration is presented as a percentage of control (no inhibitor = 0%). p24 and r.l.u. values in the absence of inhibitor were typically 15 ± 3 ng/ml and 24,500 ± 9,000, respectively. Background r.l.u. values were 7 ± 2. Each data point represents the mean ± SD of three and seven replicates for the replication and fusion assays, respectively. The horizontal dashed lines in B indicate 50% and 90% inhibition.
Figure 2
Figure 2
Effect of TAK-779 on the binding of gp120 and mAbs to CCR5. (A) The extent of gp120JR-FL binding (as a CD4-IgG2 complex) to L1.2-CCR5 cells in the absence of TAK-779 was defined as 100% (m.f.i. 40 ± 5). Binding in the presence of TAK-779 is expressed as a percentage of control. When untransfected L1.2 cells were used, binding of the gp120-CD4-IgG2 complex was negligible (<10%; m.f.i. 2 ± 1). (B) Binding of the indicated mAbs (50 nM) or gp120JR-FL (50 nM plus 50 nM of CD4-IgG2) to L1.2-CCR5 cells was measured with and without 100 nM TAK-779. The extent of mAb binding in the absence of TAK-779 was defined as 100% (m.f.i. were 50–400, depending on the mAb). Binding in the presence of TAK-779 is expressed as a percentage of control. When untransfected L1.2 cells were used, mAb binding was negligible (m.f.i. ≈2). mAbs PA8 and PA12 bind to the CCR5 Nt; 2D7 to ECL-2; PA10 and PA14 to composite epitopes involving Nt and ECL-2 (19).
Figure 3
Figure 3
Effects of alanine substitutions in CCR5 on inhibition of HIV-1JR-FL entry by TAK-779. (A) Alanine mutants of charged, polar and bulky, nonpolar amino acid residues in the Nt and ECL-1 of CCR5 were evaluated for their ability to mediate HIV-1 entry in the presence of 200 nM TAK-779. (B) The alanine mutants were located in ECL-2 and ECL-3. (C) The alanine mutants were located in the TM domain. The vertical line in each panel indicates the level of entry (14%) above which a CCR5 mutant was considered to have reduced sensitivity to TAK-779. Each data point represents the mean of three independent experiments. The different CCR5 mutants supported 10–120% of the level of entry for wild-type CCR5.
Figure 4
Figure 4
Structural model of the TM domain of the CCR5 receptor. TM helical segments, labeled 1–7, are shown as cyan-colored ribbons. The amino acid residues substituted by alanine are shown with space-filling atoms and are color-coded as follows: alanine substitutions of red-colored residues had a strong inhibitory effect on the antiviral activity of TAK-779 (Leu33, Tyr37, Trp86, Tyr108, Thr123); alanine substitutions of orange-colored residues had an intermediate effect (Arg31, Thr82, Ile198, Glu283); alanine substitutions of yellow-colored residues had a borderline effect (Phe79, Leu104); alanine substitutions of dark blue-colored residues had no effect (Phe41, Asn48, Ile52, Leu55, Ile56, Leu69, Asn71, Asp76, Thr105, Phe112, Phe113, Phe117, Phe118, Leu121, Leu122, Phe144, Thr195, Leu255, Asn258, Thr259, Met279, His289, Tyr297). Light blue-colored residues indicate mutations that caused poor expression of CCR5 (Tyr68, Phe85, Tyr251, Asn252, Asn293). These receptors could not be evaluated for HIV-1 entry. (A) View of CCR5 from within the plane of the membrane. The extracellular surface is toward the top of the figure, the cytoplasmic surface toward the bottom. For orientation, Arg31 is at the upper left in orange, and Phe144 is at the lower right in blue. (B) View of CCR5 from its extracellular surface. The model is rotated by approximately 90° from the orientation in A.
Figure 5
Figure 5
Structural models of the TAK-779 inhibitor and the CCR5 receptor. (A) Space-filling and stick representations of minimized TAK-779 structure. Atoms are color coded: carbon, green; oxygen, red; nitrogen, blue; hydrogen, gray. TAK-779 has two roughly planar segments connected by an amide bond. Its hydrophobic 4-methylphenyl ring is thought to interact with critical residues on CCR5, whereas the positively charged aminium tetrahydro-2H-pyran end of TAK-779 is oriented along the extracellular surface of CCR5, where it may block the binding of chemokine ligands and the gp120-CD4 complex. (B) The CCR5 structural model viewed from within the plane of the membrane. The CCR5 color-coding scheme is the same as in Fig. 4. Amino acid residues nearest the extracellular surface include Leu33, Trp86, and Glu283. Residues Tyr37, Thr82, and Tyr108 are deeper within the receptor. The indicated cluster of amino acids in the TAK-779 binding site includes several aromatic residues (Tyr37, Trp86, and Tyr108) that may form favorable interactions with the aromatic rings of TAK-779. (C) CCR5 viewed from the extracellular side of the receptor to show the orientation of TAK-779 binding. The colors are the same as in B, but the model is rotated by 90°. The proline residues at positions 34, 35, and 84 on TM helices 1 and 2 may facilitate the opening of the binding pocket for TAK-779. The scale is the same in A–C.
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