Understanding functional roles and regulation of granzymes

Division Of Basic Sciences - Nci

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Abstract

Our objective is to develop selective and potent substrates and probes for human granzyme A/B. We are developing selective activity-based probes to understand the role granzymes play in the tumor microenvironment. We have begun our studies examining various cell signaling pathways and transcription factors for their role in regulating the expression, secretion, and functional activity of immune cell- and tumor-derived granzymes. To generate probes, we are using a substrate-based approach where bioisosteric replacements, constrained cyclic scaffolds, and nonpeptidic fragments are incorporated into the substrate recognition element. Current approaches for measuring granzyme activity suffer from a lack of selectivity and poor kinetic properties. For example, the tetrapeptide sequence IEPD has been reported to be optimal for granzyme B (GzmB) and utilized in most of GzmB probe design. However, it shows cross-reactivity with caspase-8, limiting its utility. We have synthesized a focused library of sulfonic and phosphonic acid, triazole and tetrazole containing building blocks that are incorporated into the peptide sequence through Fmoc-based solid-phase peptide synthesis (SPPS). Initial results showed that the substitution of the P1 aspartic acid (Asp) with cysteic acid (Cya) led to an increase in substrate turnover by GzmB and more importantly, enhanced selectivity for GzmB over Casp-8 compared to the IEPD. When coupled to a serine trapping diphenylphosphonate warhead, the IEPCya sequence showed potent inhibition of GzmB activity. This polar substitution also presents a way to manipulate cellular uptake of probes targeting extracellular granzymes. We then prepared fluorescent activity-based probe (ABP) equipped with a cyanine dye at the N-terminus for labeling studies to determine the GzmB activity in cells. For in vivo imaging of GzmB activity, we are cur generating activatable (turn-ON) GzmB probes that include quenched ABPs and FRET probes as well as a near-infrared (NIR) probes that use optimized GzmB cleavable sequence. With a panel of these probes with different modalities ready, we are currently working to establish mouse tumor models in which the probe will be tested for in vivo imaging of immune cell recruitment, activation and immunotherapy responses.