Peptide length and leaving-group sterics influence potency of peptide phosphonate protease inhibitors

Abstract

The ability to follow enzyme activity in a cellular context represents a challenging technological frontier that impacts fields ranging from disease pathogenesis to epigenetics. Activity-based probes (ABPs) label the active form of an enzyme via covalent modification of catalytic residues. Here we present an analysis of parameters influencing potency of peptide phosphonate ABPs for trypsin-fold S1A proteases, an abundant and important class of enzymes with similar substrate specificities. We find that peptide length and stability influence potency more than sequence composition and present structural evidence that steric interactions at the prime-side of the substrate-binding cleft affect potency in a protease-dependent manner. We introduce guidelines for the design of peptide phosphonate ABPs and demonstrate their utility in a live-cell labeling application that specifically targets active S1A proteases at the cell surface of cancer cells.

Figure 1

General synthesis of 4-amidinophenyl-glycine that incorporates benzamidine at the P1 position. Reagents and conditions: (a) P(OPh)3, benzyl carbamate, HOAc, 1 h at rt, 1 h at 85 C; (b) dry EtOH, CHCl3, HCl in dioxane, 5 d at 4 C; (c) NH3 in dioxane, dry EtOH:dioxane (1:1), 2 d at rt; (d) H2, cat. Pd(10% on C), HCl, EtOH, 6 h at rt, (e) EDAC, HOBt, TFE:DCM, 6 h at rt. See also Table S1.

Figure 2

a: Structure of the catalytic domain of MT-SP1 bound to Benzamidine phosphonate. Numbered amino acid residues correspond to chymotrypsin protease numbering. b: Close-up ribbon of thrombin (tan) overlaid onto MT-SP1 (light blue). Thrombin L99 purple, MT-SP1 F99 blue, catalytic triad cyan. c: Thrombin (tan) surface overlaid onto MT-SP1 (light blue). Thrombin L99 purple, MT-SP1 F99 blue, catalytic triad cyan. d: Thrombin surface (tan) overlaid onto MT-SP1 (light blue) and uPA (pink). uPA His99 (yellow) occupies prime-side pocket similarly as MT-SP1 Phe99 (blue). Thrombin Leu99 purple, catalytic triad cyan. See also Figure S2.

Figure 3

Cell surface labeling of active serine proteases. PDAC2.1 and PC3 cells were imaged in the presence (3A, 3C) or absence (3B, 3D) of ABP. Cell membranes were stained with tetramethylrhodamine-conjugated wheat germ agglutinin (red), and labeled proteases were stained with streptavidin-AlexaFluor 488 (green). Colocalization of labeled proteases with membrane is seen at the cell surface and in discrete puncta, but only in the presence of ABP. Flow cytometry was used to quantify labeled streptavidin on the surface of cells (panel E). Averaged mean fluorescence intensity values are plotted normalized to background staining of labeled streptavidin in the absence of ABP. P-values correspond to a two-sample T-test, and error bars correspond to the addition of the Relative Standard Error values for the two averages multiplied by the normalized mean of each sample.