Glycopeptide analogues of PSGL-1 inhibit P-selectin in vitro and in vivo

Abstract

Blockade of P-selectin (P-sel)/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogues. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-sel with nanomolar affinity (Kd~22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-sel/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.

Figure 1. Synthesis of the Core-2 glycan and subsequent enzymatic steps to afford a family of 17 glycopeptide mimetics of PSGL-1

Enzymatic steps (a) UDP-Gal, β-1,4-GalT (bovine), alkaline phosphatase, 130 mM HEPES, pH 7.4, 40 mM sodium cacodylate, pH 7.0, 20 mM MnCl₂, and 0.02% NaN₃; (b) α 2,3-(N)-sialylT CMP-NeuAc 50 mM MOPS, pH 7.4, 0.1% bovine serum albumin, and 0.02% NaN₃, 14 h; (c) GDP-Fuc, α1,3-FucT-VI, 50 mM MOPS, pH 7.4, 20 mM MnCl₂ and 0.02% NaN₃, 16 h. The desialylated GSnP-6 was obtained from GSnP-4 in 45% yield. GSnP-7 was obtained in 55% yield by fucosylation of disialylated GSnP-6. 4-(Sulfomethyl)phenylalanine series (n): GSnP-6(X: CH₂; R=H); GSnP-7 (X: CH₂; R=Sialyl); 4-(Sulfo)phenylalanine series (n₂): GSn₂P-6 (X: bond; R=H); Tyrosine O-sulfate series: GSP-6 (X: O; R=H). The last numeral refers to the size of the glycan (e.g. 6 for hexasaccharide).

Figure 2. Binding of P-, E-, or L-selectin to PSGL-1 glycopeptide mimics and GSnP-6 stability analysis

Microarray binding studies of glycopeptide mimics toward (A) human and mouse P-selectin (5 μg/mL), (B) human and mouse L-selectin (20 μg/mL), and (C) human and mouse E-selectin (20 μg/mL). The compounds printed on the microarray in the order depicted here in A-C are detailed in Supplementary Figure 18. Reference compounds included sialyl Lewis × (sLe^x), the biantennary glycans NA2, NA2,3, NA2,6, as well as lacto-N-neo-tetraose (LNnT) and biotin. Bound selectin-Igs were detected using Alexa-488 labeled anti-human IgG antibody (5 μg/mL). Three lectins RCA-1, AAL, and PNA were used to confirm the sequence of enzymatic steps. Monoclonal antibodies CHO131, PSG2 antibody and PL-1 were used to confirm the presence of sLe^x, tyrosine sulfates, and the peptide sequence, respectively. Biacore binding analysis to human P-selectin with observed rate constants for (D) GSnP-6, k_on 3.1 × 10⁵ M⁻¹ s⁻¹, k_off 6.9 × 10³ M⁻¹s; GSn₂P-6, k_on 6.4 × 10⁵ M⁻¹s⁻¹, k_off 6.8 × 10³ M⁻¹s. Biacore binding analysis to mouse P-selectin with observed rate constants for (E) GsnP-6, k_on 4.9 × 10⁴ M⁻¹s⁻¹, k_off 8.0 × 10³ M⁻¹s⁻; GSn₂P-6 k_on 5.3 × 10⁴ M⁻¹s⁻¹, k_off 9.0 × 10³ M⁻¹s. (F) Temperature- and pH-dependent stability studies of GSnP-6.

Figure 3. Interactions of the N-terminus of PSGL-1 and GSnP-6 bound to P-selectin, as a function of the protonation state of H114

Conformation of PSGL-1 (A) and GSnP-6 (B) ligands most similar to the average shape acquired from MD simulations performed with neutral H114. The crystal structure of the PSGL-1 ligand is shown in red with a splined representation of the peptide backbone, sulfated amino acid positions in green, and a stick representation for monosaccharide rings. Assuming that H114 is fully protonated leads to optimal reproduction of the crystallographic data for PSGL-1 (C) and leads to similar binding for GSnP-6 (D). The solvent-accessible surface of P-selectin is colored according to the electrostatic potential (acidic region: red; basic region: blue). (E-G) Hydrogen bonds between Fuc (red), Core-2 Gal (yellow), and Neu5Ac (purple) and P-selectin residues.

Figure 4. GSnP-6 inhibits selectin adhesive interactions in vitro and in vivo

(A-B) GSnP-6 (0 - 30 μM) was incubated with (A) human circulating PMN and monocytes or (B) mouse circulating PMN and monocytes and species appropriate P-selectin chimera, analyzed by flow cytometry and plotted as percent inhibition vs. PBS control. GSnP-6 inhibited P-selectin dependent interactions in a dose dependent manner in human and mouse leukocytes, including human neutrophils (IC₅₀ ~ 14 μM), human monocytes (IC₅₀ ~ 20 μM), mouse neutrophils (IC₅₀ ~ 19 μM), and mouse monocytes (IC₅₀ ~ 28 μM). (C) GSnP-6 inhibited PSGL-1/L-selectin interactions to human U937 and mouse neutrophils with a lower potency (IC₅₀ ~ 30 μM). Data are mean ± SEM, n=3. (D-F) GSnP-6 inhibits P-selectin/PSGL-1 adhesion under shear in vitro. (D) GSnP-6 (0-6 μM) exhibits dose dependent inhibition of human neutrophil and human monocyte rolling and arrest on a recombinant ICAM-1/P-selectin substrate under shear; n=3 PBS, n=3/GSnP-6 dose, velocities (μm/s) of 120 cells per condition were measured, data are plotted as mean ± SEM. (E-F) GSnP-6 increases rolling velocity of neutrophils and monocytes, cumulative frequency histograms are shown for PBS vehicle control (E) and 2 μM GSnP-6 (F); Vneutrophil: saline vs. GSnP-6, p < 0.0001 (Student's t-test); Vmonocyte: saline vs. GSnP-6, p < 0.0001 (Student's t-test). (G-I) GSnP-6 (4 μmol/kg, n=4 mice, 5 leukocytes/vessel analyzed in 28 vessels) or saline control (n=3 mice, 5 leukocytes/vessel analyzed in 21 vessels) were delivered IV and leukocyte rolling velocity was recorded 15-25 min post-surgical stimulation of the mouse cremaster to characterize P-selectin dependent responses in vivo; Vmean: saline vs. GSnP-6, p < 0.01 (Student's t-test). (I) Cumulative frequency histogram of leukocyte rolling velocities, median values indicated by vertical lines (median velocity saline = 35.5 μm/s, median velocity GSnP-6 = 64.0 μm/s).

Figure 5. GSnP-6 limits platelet-leukocyte aggregation in vitro and in vivo

(A-D) Anti-coagulated human or mouse blood was dosed with 40 μM GSnP-6 at room temperature and stimulated for platelet P-selectin expression with thrombin receptor-activating peptide (40 μM human PAR₁-activating peptide, 200 μM mouse PAR₄-activating peptide). Platelet-leukocyte aggregates were quantified by two-color flow cytometry, CD45⁺ monocyte and neutrophil populations were discerned through characteristic side scatter as quantified as % platelet positive in saline control, 40 μM GSnP-6, and anti-CD62P (5 μg/mL human KPL-1, 5 μg/mL mouse RB40.34) treated samples. (A) Representative scatter plot of human samples incubated with anti-CD42a-PE and anti-CD45-APC, (B) % platelet positive neutrophils and monocytes in human samples, GSnP-6 inhibited 65% of platelet-neutrophil and 72% of platelet-monocyte aggregate formation in human blood. (C) Representative scatter plot of mouse samples incubated with anti-CD41-PE and anti-CD45-APC, (D) % platelet positive neutrophils and monocytes in mouse samples, GSnP-6 inhibited 42% of platelet-neutrophil and 47% of platelet-monocyte aggregate formation in mouse blood. Data representative of triplicate sample mean ± SEM, *p<0.05 vs. saline control (Student's t-test). (E-G) In vivo platelet-leukocyte aggregation in a TNF-α model of venular inflammation. (E) Intravital microscopy of venular inflammation 3 hr after administration of TNF-α demonstrates platelet aggregation and platelet-PMN binding after administration of saline control (PMN green; platelet red). (F) Platelet aggregation and platelet-PMN binding are not observed after administration of GSnP-6 (4 μmol/kg IV). (G) The platelet inhibitory effect of GSnP-6 was equivalent to that observed for anti-CD62P, a P-selectin blocking antibody (75 μg/mouse IV) and was significantly less than saline control (*p < 0.05 vs. saline (Student's t-test), n=5 mice/treatment, 8-10 venules/mouse analyzed; error bars are SEM).