Spirodiketopiperazine-based CCR5 inhibitor which preserves CC-chemokine/CCR5 interactions and exerts potent activity against R5 human immunodeficiency virus type 1 in vitro

Abstract

We identified a novel spirodiketopiperazine (SDP) derivative, AK602/ONO4128/GW873140, which specifically blocked the binding of macrophage inflammatory protein 1alpha (MIP-1alpha) to CCR5 with a high affinity (K(d) of approximately 3 nM), potently blocked human immunodeficiency virus type 1 (HIV-1) gp120/CCR5 binding and exerted potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates, including multidrug-resistant HIV-1 (HIV-1(MDR)) (50% inhibitory concentration values of 0.1 to 0.6 nM) in vitro. AK602 competitively blocked the binding to CCR5 expressed on Chinese hamster ovary cells of two monoclonal antibodies, 45523, directed against multidomain epitopes of CCR5, and 45531, specific against the C-terminal half of the second extracellular loop (ECL2B) of CCR5. AK602, despite its much greater anti-HIV-1 activity than other previously published CCR5 inhibitors, including TAK-779 and SCH-C, preserved RANTES (regulated on activation normal T-cell expressed and secreted) and MIP-1beta binding to CCR5(+) cells and their functions, including CC-chemokine-induced chemotaxis and CCR5 internalization, while TAK-779 and SCH-C fully blocked the CC-chemokine/CCR5 interactions. Pharmacokinetic studies revealed favorable oral bioavailability in rodents. These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection.

FIG. 1.

Structures of AK602 and AK530.

FIG. 2.

Variability of anti-HIV-1 activity of AK602 in phytohemagglutinin-peripheral blood mononuclear cells. The range of IC₅₀ values of E913 and E921/TAK-779 against HIV-1_Ba-L varied substantially when examined in multiple phytohemagglutinin-peripheral blood mononuclear cells as target cells, 14 to 650 nM (n = 11) and 2 to 200 nM (n = 15), respectively, while that of AK602 was relatively narrow, 0.1 to 1 nM (n = 15), similar to that of zidovudine (ZDV), 1 to 9 nM (n = 14).

FIG. 3.

CCR5 binding profiles and rgp120 binding blocking of various CCR5 inhibitors. (A) Binding affinity of AK602 to CCR5. CCR5⁺ CHO cells were incubated with the ³H-labeled CCR5 inhibitors AK530, AK602, E913, E921/TAK-779, and AK671/SCH-C for 1 h. Following thorough washing, cells were lysed, the radioactivity in the lysates was determined, and B_max and K_d values were calculated. The K_d values thus obtained were 0.4 ± 0.4, 2.9 ± 1.0, 111.7 ± 3.5, 32.2 ± 9.6, and 16.0 ± 1.5 nM, respectively. All assays were independently performed 3 to 10 times, and the values represent the arithmetic means ± 1 standard deviation. (B) AK602 potently blocks the binding of rgp120/sCD4 to CCR5. CCR5⁺ CHO cells were incubated with rgp120 (5 μg/ml) and sCD4 (5 μg/ml) in the presence or absence of the indicated concentrations of CCR5 inhibitors, and the binding of rgp120/sCD4 complex to CCR5⁺ CHO cells was determined. The 50% binding inhibition (EC₅₀) value was determined based on the mean fluorescence intensity values obtained with or without CCR5 inhibitors. EC₅₀ values for AK602, AK530, E921/TAK-779, and AK671/SCH-C were 2.7, 280, 12.0, and 16.5 nM, respectively.

FIG. 4.

AK602 binds to the second extracellular loop of CCR5. AK602 at 100 nM almost completely inhibited the binding of two monoclonal antibodies, 45523, directed against multidomain epitopes of CCR5, and 45531, recognizing ECL2B of CCR5. In contrast, E921/TAK-779 and AK671/SCH-C moderately blocked the binding of 45523 and 45531. Note that there was no AK602 inhibition of the binding of a monoclonal antibody 2D7, which is known to bind to domain A of ECL2 of CCR5.

FIG. 5.

Inhibition of CC-chemokine binding to CCR5 by various CCR5 inhibitors. CCR5⁺ CHO cells were incubated with 3 nM [¹²⁵I]RANTES (A), [¹²⁵I]MIP 1 β (B), or [¹²⁵I]MIP-1 α (Pnel C) in the presence and absence of various concentrations of CCR5 inhibitors. Note that while AK671/SCH-C and E921/TAK-779 completely inhibited the binding of [¹²⁵I]RANTES, [¹²⁵I]MIP-1α, and [¹²⁵I]MIP-1β to CCR5, SDP derivatives partially blocked RANTES (A) and MIP-1β (B) binding, although they completely blocked MIP-1α binding (C).

FIG. 6.

AK602 and RANTES bind simultaneously to CCR5. (A) CCR5⁺ CHO cells were exposed to 10 nM [³H]AK602 and various concentrations of unlabeled RANTES. After 1 h of incubation, the cells were washed, and the [³H]AK602 bound to the cells was measured. Note that 100% radioactivity on the ordinate denotes the radioactivity of cell-bound [³H]AK602 without RANTES and that ≈90% of CCR5 molecules are bound to AK602 at 10 nM (Fig. 3A). (B) CCR5⁺ CHO cells were exposed to 10 nM unlabeled AK602 and various concentrations of [¹²⁵I]RANTES. After 1 h of incubation, the cells were washed, and the [¹²⁵I]RANTES bound to the cells was measured. The binding profile of [¹²⁵I]RANTES alone is illustrated by open circles. Note that 100% radioactivity is equated to the radioactivity of cell-bound [¹²⁵I]RANTES at 10 nM. The K_d values of RANTES in the presence and absence of 10 nM AK602 were 4.5 and 0.6 nM, respectively.

FIG. 7.

AK602 allows RANTES-induced chemotaxis and CCR5 internalization. (A) CCR5⁺ MOLT4 cells were exposed to various concentrations of AK530, AK602, E921/TAK-779, or AK671/SCH-C, thoroughly washed, plated onto the upper chamber of the ChemTx System, exposed to 0.5 nM RANTES contained in the lower chamber, and incubated for 4 h; the number of the cells which migrated to the lower chamber was determined, and chemotaxis was calculated. (B) CCR5⁺ CHO cells were exposed to 10 nM RANTES in the presence or absence of various concentrations of each CCR5 inhibitor and washed with acidic solution for removal of the cell-bound RANTES (21). The amount of cell surface CCR5 was subsequently determined with monoclonal antibody 3A9 (BD PharMingen), which recognizes the N terminus of CCR5 and competes with none of the CCR5 inhibitors tested. In panel A, the level of chemotaxis suppression by TAK-779 and SCH C was greater than that by AK530 and AK602 at four concentrations examined, although complete suppression was seen only at the highest concentration of the AK compounds, 1 μM. However, in panel B, the level of CCR5 internalization suppression by TAK-779 and SCH-C was greater than that of the AK compounds at all three concentrations examined.