Small Molecule Inhibitors of Lymphocyte Perforin as Focused Immunosuppressants for Infection and Autoimmunity

Abstract

New drugs that precisely target the immune mechanisms critical for cytotoxic T lymphocyte (CTL) and natural killer (NK) cell driven pathologies are desperately needed. In this perspective, we explore the cytolytic protein perforin as a target for therapeutic intervention. Perforin plays an indispensable role in CTL/NK killing and controls a range of immune pathologies, while being encoded by a single copy gene with no redundancy of function. An immunosuppressant targeting this protein would provide the first-ever therapy focused specifically on one of the principal cell death pathways contributing to allotransplant rejection and underpinning multiple autoimmune and postinfectious diseases. No drugs that selectively block perforin-dependent cell death are currently in clinical use, so this perspective will review published novel small molecule inhibitors, concluding with in vivo proof-of-concept experiments performed in mouse models of perforin-mediated immune pathologies that provide a potential pathway toward a clinically useful therapeutic agent.

Figure 1

Mechanism of perforin-dependent cell killing. Formation of an immune synapse between a cytotoxic lymphocyte and a target cell results in granzymes (black dots) entering the target cells through perforin pores (light blue) in the target cell plasma membrane. Also shown is the microtubule-organizing center that enables cytotoxic granule polarization to the cell membrane where perforin and the granzymes are released into the synapse. The lymphocyte is protected from the secreted perforin by high lipid order domains (red) and exposed phosphatidylserine (green) at the immunological synapse. Reprinted with permission from ref (5). Copyright 2019 Springer Nature.

Figure 2

Examples of V-ATPase inhibitors.

Figure 3

Top three validated hits from the high-throughput screen.

Figure 4

Summary of dihydrofuro[3,4-c]pyridine structure–activity relationship (SAR) and preferred analogue 9.

Figure 5

Summary of amino-2,3-dicyanopyrido[1,2-a]benzimidazole SAR and preferred analogues 10 and 11.

Figure 6

Summary of aryl-substituted isobenzofuranone SAR and preferred analogues 12, 13, and 14.

Figure 7

Summary of diarylthiophene SAR and preferred analogues 15 and 16.

Figure 8

Summary of initial benzenesulfonamide strategy and preferred analogue 18.

Figure 9

Summary of SAR for benzenesulfonamide and pyridine and preferred analogues 26 and 27.

Figure 10

Summary of SAR for aza-isoindolinones and preferred analogue 28.

Figure 11

Examples of parent and prodrug perforin inhibitors.

Figure 12

Schematic illustration of pharmacokinetics and targeting properties of the LAT1-utilizing prodrug of perforin inhibitor. The prodrugs can be accumulated via LAT1 into the (A) brain and (B) pancreas. In the brain, the LAT1-utilizing prodrugs can further accumulate into the (C) cytolytic effector cells, or brain parenchymal cells (expressing LAT1), and intracellularly into the (D) cytolytic granules or lysosomes, in which the prodrug is bioconverted to release the active perforin inhibitor. The perforin inhibitor itself cannot be delivered into the brain, although it can be accumulated into slightly acidic organelles most likely due to the pH difference.

Figure 13

Compound 27 mouse pharmacokinetics and efficacy. (A) Plasma concentration–time profiles of multiple dose levels of 27 by single dose. (B) Simulated plasma concentration–time profiles of different multiple dose schedules of 27 based on a one-compartment pharmacokinetic model. (C) Pharmacokinetic/pharmacodynamic relationship for compound 27 that showed the strongest PK/PD correlation was percent perforin inhibition in vivo (peripheral blood) and the time that total plasma concentrations remained above 900 μM. Modified from ref (108). Copyright 2022 American Chemical Society.