Calpain-2 Inhibitors as Therapy for Traumatic Brain Injury

Abstract

While calpains have long been implicated in neurodegeneration, no calpain inhibitor has been developed for the treatment of neurodegeneration. This is partly due to the lack of understanding of the specific functions of most of the 15 members of the calpain family. Work from our laboratory over the last 5-10 years has revealed that calpain-1 and calpain-2, two of the major calpain isoforms in the brain, play opposite roles in both synaptic plasticity/learning and memory and neuroprotection/neurodegeneration. Thus, calpain-1 activation is required for triggering certain forms of synaptic plasticity and for learning some types of information and is neuroprotective. In contrast, calpain-2 activation limits the extent of synaptic plasticity and of learning and is neurodegenerative. These results have been validated with the use of calpain-1 knock-out mice and mice with a selective calpain-2 deletion in excitatory neurons of the forebrain. Through a medicinal chemistry campaign, we have identified a number of selective calpain-2 inhibitors and shown that these inhibitors do facilitate learning of certain tasks and are neuroprotective in a number of animal models of acute neurodegeneration. One of these inhibitors, NA-184, is currently being developed for the treatment of traumatic brain injury, and clinical trials are being planned.

Keywords: Biomarker; Calpain; Concussion; Learning and memory; Neurodegeneration.

Fig. 1

Schematic diagram summarizing the opposite functions of calpain-1 and calpain-2 following TBI. Calpain-1 is rapidly but transiently activated following TBI. Its activation results in the stimulation of neuroprotective pathways, including the Akt pathway. On the other hand, calpain-2 activation is delayed and prolonged lasting for several days after TBI. Its activation leads to the stimulation of neurodegenerative pathways, including STEP and p38. In both cases, spectrin is cleaved, generating a specific breakdown product (SBDP), which is widely used as an index of brain calpain activation. We are also postulating that calpain-1 is located postsynaptically downstream of the NMDA receptors, while calpain-2 is mostly located extrasynaptically. This differential localization of calpain-1 and calpain-2 is most likely due to the existence of different PDZ binding domains in calpain-1 and calpain-2 [22]

Fig. 2

Lesion volume following TBI in wild-type, calpain-1 KO, and conditional calpain-2 KO mice. Wild-type (WT), calpain-1 knock-out (C1-KO), and conditional calpain-2 knock-out (CC2-KO) mice were subjected to the CCI model of TBI in separate experiments. Lesion volume was measured 3 days after TBI. Lesion area was measured in 8 brain sections from each animal. Total lesion volume was then measured by integrating the areas across all sections. Results are expressed as percent of the lesion volume in WT mice in the respective experiments and are means ± SEM of 8, 4, and 6 animals, respectively. *p < 0.05, as compared to WT mice. Data for the C1-KO mice are from [26] and for CC2-KO from [42]

Fig. 3

Comparison of C2I/NA-101 with other calpain inhibitors. C2I shares a similar scaffold with the other compounds shown in the figure. SNJ-1945, ABT-957, 4a, and 4d, all incorporate α-ketoamide warheads. SNJ-1945 has been reported as BBB permeable through LC–MS/MS [58]; ABT-957 was shown to cross rat BBB after intraperitoneal injection and was in clinical trial phase I for AD [59, 60]; and both 4a and 4d have therapeutically useful concentrations in mouse brain after subcutaneous administration [61]. An E-64 derivative has been reported to effectively penetrate the brain and significantly inhibit calpain-catalyzed hydrolysis of spectrin [62]. However, none of these calpain inhibitors shows calpain-2 selectivity, except C2I. Also shown is the location of the two chiral centers and the docking of the two diastereoisomers, S,S-C2I and S,R-C2I, in the catalytic site of calpain-2, with their respective Ki against calpain-1 (cal-1) or calpain-2 (cal-2)

Fig. 4

Comparison between SILCS FragMaps of calpain-1 (a, c) and calpain-2 (b, d). Green mesh density is polar FragMap, blue is hydrogen bond donor FragMap, and red is hydrogen bond acceptor FragMap. The cutoff value for all density mesh is set to be − 1. Yellow circles indicate that additional polar group is favored at para-position in calpain-1 P1’ site (a), but at meta-position in calpain-2 P1’ site (b); no density was found at ortho-position in calpain-1 P1’ site (c), but hydrogen bond donor and acceptor densities were present at ortho-position in calpain-2 (d, yellow circled area)

Fig. 5

Structure of the selective calpain-2 inhibitors NA-112 and NA-184