Allosteric Tuning of Caspase-7: A Fragment-Based Drug Discovery Approach

Abstract

The caspase family of cysteine proteases are highly sought-after drug targets owing to their essential roles in apoptosis, proliferation, and inflammation pathways. High-throughput screening efforts to discover inhibitors have gained little traction. Fragment-based screening has emerged as a powerful approach for the discovery of innovative drug leads. This method has become a central facet of drug discovery campaigns in the pharmaceutical industry and academia. A fragment-based drug discovery campaign against human caspase-7 resulted in the discovery of a novel series of allosteric inhibitors. An X-ray crystal structure of caspase-7 bound to a fragment hit and a thorough kinetic characterization of a zymogenic form of the enzyme were used to investigate the allosteric mechanism of inhibition. This work further advances our understanding of the mechanisms of allosteric control of this class of pharmaceutically relevant enzymes, and provides a new path forward for drug discovery efforts.

Keywords: allostery; apoptosis; caspases; drug discovery; enzyme catalysis.

Figure 1

A) Chemical structures of 1 and 2, identified from fragment screening. B) Michaelis–Menten kinetics. In the presence of increasing concentrations of 1, there is an apparent lowering of k _cat, while K _m is relatively constant. This is consistent with a non‐competitive mechanism of inhibition. C) DSF of C7 in the presence of DMSO (E), 1 (EI), substrate Ac‐VAD‐FMK (ES), and both 1 and Z‐VAD‐FMK (ESI) shown in the bar graph on the right in triplicate. D) Binding of 1 to C7 as measured by SPR. OneStep injection was fit with a simple 1:1 binding model shown in red, raw data are shown in black. Further details on the OneStep injection and analysis can be found in the Supporting Information.

Figure 2

A) Crystal structure of C7 soaked with 1 (PDB ID: 5V6U), polder F _o−F _c omit map contoured at 5.0 σ r.m.s. (root mean square) and carved 5 Å around 1 (cyan sticks) at the dimer interface; neighboring residues (<8 Å) are shown as green sticks and water molecules as red spheres. B) Alignment of catalytic C186/H144, shown in sticks, for mature C7 (4FDL, cyan) and C7 bound to 1 (5V6U, light gray/white). C) Overview of the C7 dimer bound to substrate (PDB ID: 1F1J). Colors highlight monomer A (blue), monomer B (cyan), peptide substrate Ac‐DEVD‐CHO (green sticks), and the allosteric site (gray surface). D) Close‐up view of the active‐site loop bundle. The Ac‐DEVD‐CHO peptide substrate is shown as green sticks, loops are colored red (L1), orange (L3), yellow (L4), and magenta (L2/L2′), and L2′ is donated from the opposite monomer. Cartoon representation of E) active C7 and F) C7 bound to 1 (cyan sticks; 5V6U), colored according to their normalized B‐factors from low to high (blue to red). Dark gray loops for 5V6U represent disordered regions not modelled into the crystal structure and are overlaid from 4FDL for reference.