Calpain research for drug discovery: challenges and potential

Key Points

  • The calpains are a family of proteases with biologically vital functions. However, the mechanistic features of calpains are largely unknown.

  • Calpains have been identified as potential therapeutic targets for various types of diseases, including neurodegenerative and cardiovascular disorders, ophthalmic diseases and cancer.

  • Many disease phenotypes are ameliorated by calpain inhibition, and some calpain inhibitors have entered clinical trials.

  • Many calpain orthologues in parasites or microorganisms are responsible for the pathogenicity and viability of the organism; thus, targeting these calpains is a promising approach for combatting infectious diseases.

  • Some calpain gene defects resulting in loss of calpain activity are pathologically implicated in human disease. Therefore, in addition to therapies that inhibit calpain activity, developing strategies that compensate for calpain loss are an important goal.

  • The development of inhibitors with improved efficiency and specificity for calpains is a critical future research direction. Unveiling the physiological functions of calpains at the molecular level is a key challenge.

Abstract

Calpains are a family of proteases that were scientifically recognized earlier than proteasomes and caspases, but remain enigmatic. However, they are known to participate in a multitude of physiological and pathological processes, performing 'limited proteolysis' whereby they do not destroy but rather modulate the functions of their substrates. Calpains are therefore referred to as 'modulator proteases'. Multidisciplinary research on calpains has begun to elucidate their involvement in pathophysiological mechanisms. Therapeutic strategies targeting malfunctions of calpains have been developed, driven primarily by improvements in the specificity and bioavailability of calpain inhibitors. Here, we review the calpain superfamily and calpain-related disorders, and discuss emerging calpain-targeted therapeutic strategies.

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Figure 1: Schematic structure of calpains.
Figure 2: Cross-eye stereo view of CAPN1, 2, 8 and 9.

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Acknowledgements

The authors thank all of the laboratory members for their invaluable support, Y. Ogata for thoroughly surveying the clinical trial studies of calpain-related agents, S. Ishiura for valuable advice on early clinical studies of calpain inhibitors, and L. Miglietta and G. Gray for their excellent English editing. This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (JP25440059 to Y.O. and JP15H02389 to H.S.), Open Partnership Joint Projects of the JSPS Bilateral Joint Research Projects (to H.S.), a Takeda Science Foundation research grant (to Y.O. and H.S.), the Council for Science, Technology and Innovation's Strategic Innovation Promotion Programme “Technologies for creating next-generation agriculture, forestry, and fisheries” (funded by BTRAI-NARO to H.S.), and a research grant of The Naito Foundation (to H.S.).

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Glossary

Clan

Peptidases and their proteinaceous inhibitors are systematically classified by the online MEROPS database. A clan is composed of multiple families, each of which corresponds to a group of orthologous peptidases. Members of a clan share similar primary and tertiary structures. This classification system complements and extends the previously established enzyme classification system, which includes all enzymes.

CysPc motif

A protease catalytic domain of calpains. CysPc-containing proteases belong to the same clan as papain, but unlike the catalytic domain of papain the CysPc divides into two separate structures, protease core 1 (PC1) and PC2, in the absence of Ca2+. Amino acid sequence comparisons suggest that all of the calpain species for which 3D structures are not yet solved share similarity in their CysPc domain.

α-Ketoamide inhibitor

A class of reversible inhibitors for cysteine, serine or threonine proteases that add an electrophile to these active site amino acid residues. Many inhibitors of this class have been developed by systematically replacing aldehyde moieties of known calpain inhibitors with α-ketoamide, and subsequently modifying other positions to improve effectiveness.

Calpainopathies

Diseases caused by a genetic defect in a calpain gene. The pathogenic mechanism can be either loss of function (for example, limb-girdle muscular dystrophy type 2A is caused by inactivating mutations in the CAPN3 gene) or gain of function (for example, autosomal dominant neovascular inflammatory vitreoretinopathy is caused by an excessive activation of CAPN5 due to mutations).

Calpain-type β-sandwich

(CBSW). A domain whose 3D structure, but not primary sequence, shows overall similarity to the C2 domain, a calcium-binding motif found in protein kinase C, synaptotagmins and other calcium-related proteins. The CBSW domain has a role in substrate recognition.

Penta-EF-hand

(PEF). Among the proteins with Ca2+-binding EF-hand motifs, those with five EF-hand motifs in tandem comprise the PEF family. The fifth EF region is often involved in homo- or heterodimerization. Classical calpains have a PEF domain, and hence also belong to the PEF family.

CAPNS1

(Calpain small subunit 1). A PEF family member. A paralogue called CAPNS2 has an unknown function. In vivo, CAPNS1 is an essential component of the two conventional calpains, calpain-1 and calpain-2. However, in vitro, the cysteine protease domains isolated from CAPN1 and CAPN2 (mini-calpains) are functional as active proteases.

Macrocyclic inhibitor

Macrocyclic structures introduced into linear peptides or compounds often improve potency by providing well-defined conformations, such as α-helices and β-strands in the case of peptides, thereby facilitating interactions with their targets, which include proteases. This strategy has been used to develop improved calpain inhibitors.

Neglected tropical diseases

Among various parasitic and infectious diseases, 17 diseases have been recognized by the World Health Organization as targets for which control would promote an exodus from poverty somewhere in the world.

Intrinsically unstructured protein

A protein that does not possess a fixed or stable 3D structure (also called an 'intrinsically disordered protein'). Some of these proteins remain unstructured even in their functional state, whereas others adopt a fixed structure after binding to another protein. For example, calpastatin has tandemly repeated calpain inhibitory sequences, neither of which assumes a defined structure unless calpastatin is bound to calpain.

Intermolecular complementation

(iMOC). A phenomenon in which single-polypeptide-derived fragments, none of which are capable of expressing the activity of the original protein, reconstitute the original activity through spontaneous and noncovalent interaction under physiological conditions. In this process, the amino acids essential for the activity are provided by different fragments.

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Ono, Y., Saido, T. & Sorimachi, H. Calpain research for drug discovery: challenges and potential. Nat Rev Drug Discov 15, 854–876 (2016). https://doi.org/10.1038/nrd.2016.212

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