Calpain Inhibitors as Potential Therapeutic Modulators in Neurodegenerative Diseases.

It is considered a significant challenge to understand the neuronal cell death mechanisms with a suitable cure for neurodegenerative disorders in the coming years. Calpains are one of the best-considered "cysteine proteases activated" in brain disorders. Calpain is an important marker and mediator in the pathophysiology of neurodegeneration. Calpain activation being the essential neurodegenerative factor causing apoptotic machinery activation, it is crucial to develop reliable and effective approaches to prevent calpain-mediated apoptosis in degenerating neurons. It has been recently seen that the "inhibition of calpain activation" has appeared as a possible therapeutic target for managing neurodegenerative diseases. A systematic literature review of PubMed, Medline, Bentham, Scopus, and EMBASE (Elsevier) databases was conducted. The present article reviews the basic pathobiology and role of selective calpain inhibitors used in various neurodegenerative diseases as a therapeutic target.

Tripeptide analogues of MG132 as protease inhibitors.

The 26S proteasome and calpain are linked to a number of important human diseases. Here, we report a series of analogues of the prototypical tripeptide aldehyde inhibitor MG132 that show a unique combination of high activity and selectivity for calpains over proteasome. Tripeptide aldehydes (1-3) with an aromatic P3 substituent show enhanced activity and selectivity against ovine calpain 2 relative to chymotrypsin-like activity of proteasome. Docking studies reveal the key contacts between inhibitors and calpain to confirm the importance of the S3 pocket with respect to selectivity between calpains 1 and 2 and the proteasome.

In vitro selection of Phytomonas serpens cells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps.

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

Susceptibility of promastigotes and intracellular amastigotes from distinct Leishmania species to the calpain inhibitor MDL28170.

Despite the available drug options, leishmaniasis treatment remains unsatisfactory. The repurposing of calpain inhibitors originally developed for human diseases became an interesting alternative, since Leishmania cells express calpain-related proteins. The susceptibility of six Leishmania species (L. amazonensis, L. braziliensis, L. major, L. mexicana, L. chagasi, and L. donovani) to the calpain inhibitor MDL28170 was determined. Promastigote and intracellular amastigote viability in the presence of MDL28170 was evaluated. MDL28170 was able to reduce promastigote proliferation in a dose-dependent manner for all the parasites. A significant reduction on the general parasite metabolism was detected, as judged by resazurin assay, as well as induced important morphological alterations, including rounding promastigotes and loss of the flagellum. MDL28170 was also able to reduce the number of intracellular amastigotes in RAW macrophages. The susceptibility of both parasite stages (promastigotes and amastigotes) to MDL28170 was similar for all Leishmania species tested. MDL28170 showed a much higher toxicity to Leishmania amastigotes when compared with mammalian macrophages, displaying selectivity index values varying from 13.1 to 39.8. These results suggest that the development of calpain inhibitors may represent an interesting alternative in the treatment of leishmaniasis.

Why calpain inhibitors are interesting leading compounds to search for new therapeutic options to treat leishmaniasis?

Leishmaniasis is a neglected disease, which needs improvements in drug development, mainly due to the toxicity, parasite resistance and low compliance of patients to treatment. Therefore, the development of new chemotherapeutic compounds is an urgent need. This opinion article will briefly highlight the feasible use of calpain inhibitors as leading compounds to search for new therapeutic options to treat leishmaniasis. The milestone of this approach is to take advantage on the myriad of inhibitors developed against calpains, some of which are in advanced clinical trials. The deregulated activity of these enzymes is associated with several pathologies, such as strokes, diabetes and Parkinson's disease, to name a few. In Leishmania, calpain upregulation has been associated to drug resistance and virulence. Whereas the difficulties in developing new drugs for neglected diseases are more economical than biotechnological, repurposing approach with compounds already approved for clinical use by the regulatory agencies can be an interesting shortcut to a successful chemotherapeutic treatment for leishmaniasis.

Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L.

The main protease (Mpro) of SARS-CoV-2, the pathogen responsible for the COVID-19 pandemic, is a key antiviral drug target. While most SARS-CoV-2 Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently discovered several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II/XII, which are also active against human cathepsin L, a host-protease that is important for viral entry. To determine the binding mode of these calpain inhibitors and establish a structure-activity relationship, we solved X-ray crystal structures of Mpro in complex with calpain inhibitors II and XII, and three analogues of GC-376, one of the most potent Mpro inhibitors in vitro. The structure of Mpro with calpain inhibitor II confirmed the S1 pocket of Mpro can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. Interestingly, the structure of calpain inhibitor XII revealed an unexpected, inverted binding pose where the P1' pyridine inserts in the S1 pocket and the P1 norvaline is positioned in the S1' pocket. The overall conformation is semi-helical, wrapping around the catalytic core, in contrast to the extended conformation of other peptidomimetic inhibitors. Additionally, the structures of three GC-376 analogues UAWJ246, UAWJ247, and UAWJ248 provide insight to the sidechain preference of the S1', S2, S3 and S4 pockets, and the superior cell-based activity of the aldehyde warhead compared with the α-ketoamide. Taken together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of Mpro inhibitors as SARS-CoV-2 antivirals.

An update on the therapeutic potential of calpain inhibitors: a patent review.

INTRODUCTION: Calpain is a cytosolic proteinase that regulates of a wide range of physiological functions. The enzyme has been implicated in various pathological conditions including neurodegenerative disorders, cardiovascular disorders, cancer, and several other diseases. Therefore, calpain inhibitors are of interest as therapeutic agents and have been studied in preclinical models of several diseases in which the enzyme has been implicated. AREAS COVERED: Calpain inhibitors that were disclosed over the last 5 years (2015-2019) include calpastatin-based peptidomimetics; thalassospiramide lipopeptides; disulfide analogs of alpha-mercaptoacrylic acids; allosteric modulators; azoloimidazolidenones; and macrocyclic/non-macrocyclic carboxamides. The effectiveness of some of the inhibitors in preclinical animal models is discussed. EXPERT OPINION: Significant milestones that were made over this time frame include: a) disclosure of novel blood-brain barrier (BBB) permeable calpastatin analogs as calpain inhibitors; b) disclosure that potent calpain inhibitors can be obtained by targeting the hydrophobic pockets on chain A of PEF(S) of the small subunit of calpain; c) use of PEF(S) (PDB ID: 4WQ2) in virtual screening to identify novel structurally diverse calpain inhibitors; and d) mitigation of the metabolic instability of the alpha-ketoamide warhead of calpain inhibitors.

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease.

A novel coronavirus SARS-CoV-2, also called novel coronavirus 2019 (nCoV-19), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.96% as of May 4, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three monomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.

Experimental Manipulation of Calpain Activity In Vitro.

The calpain activity in cells can be experimentally manipulated in vitro by calpain inhibitors, and various types of calpain inhibitors such as peptide aldehydes and α-mercapto-acrylic acid derivatives are widely used as a valuable tool to elucidate the physiological and pathological roles of calpain. Here I describe the experimental procedures with calpain inhibitors, with human neutrophils being primarily used in this experiment. It should be noted that potent calpain inhibitors not only inhibit the calpain activity but also stimulate cell functions via direct activation of human formyl peptide receptors and/or other G protein-coupled receptors depending on the inhibitors used.

Molecular dynamic simulations of the catalytic subunit of calpains 1, 2, 5, and 10: Structural analysis with an aim toward drug design.

Calpains are cysteine proteases involved in the development of several human chronic illnesses such as neurodegenerative diseases, cardiovascular ailments, diabetes, and obesity which constitutes them into possible therapeutic targets. Here, using molecular dynamic simulations and docking, we studied the binding of known inhibitors to representative members of classical and nonclassical calpains. Our aim is to gain better understanding on the inhibition mechanism of calpains and to develop better and more specific inhibitors. Our atomistic models confirmed the importance of calcium ions for the structure of calpains and, as a consequence, their functionality. With these models and their subsequent use in molecular docking, essential structural requirements were identified for the binding of ligands to the calpain catalytic site that provide useful information for the design of new selective calpain inhibitors.

Discovery of potent calpain inhibitors based on the azolo-imidazolidenone scaffold.

A series of new azolopyrimidine-peptide hybrids and indolomethylideneimidazolones were obtained and evaluated as calpain inhibitors. The hybrid compounds were inactive, whereas some members of the initial azolomethylideneimidazolone series showed interesting calpain inhibitory activity. By using 4b as a hit compound, a new series of analogs were synthesized by an efficient synthetic procedure based on a multicomponent reaction followed by an unprecedented reaction at the methylene position of the molecule. The best inhibitor found for calpain I (IC50 = 20 nM) was about 20 times more potent than the hit compound. Studies on 4b showed that its inhibition is consistent with an uncompetitive inhibition mode. This compound did not exhibit cellular toxicity at any of the doses tested (0.1-10 µM) and further studies indicated that it was capable of blockading chemical ischemia induction of apoptosis by preventing sodium azide-dependent calpain activation in intact human kidney tubular epithelial cells. The results of molecular modeling studies rationalized the inhibitory activity found for this series and account, from a structural point of view, for the most active compound identified (4j).

Neuroprotective strategies against calpain-mediated neurodegeneration.

Calpains are calcium-dependent proteolytic enzymes that have deleterious effects on neurons upon their pathological over-activation. According to the results of numerous studies to date, there is no doubt that abnormal calpain activation triggers activation and progression of apoptotic processes in neurodegeneration, leading to neuronal death. Thus, it is very crucial to unravel all the aspects of calpain-mediated neurodegeneration in order to protect neurons through eliminating or at least minimizing its lethal effects. Protecting neurons against calpain-activated apoptosis basically requires developing effective, reliable, and most importantly, therapeutically applicable approaches to succeed. From this aspect, the most significant studies focusing on preventing calpain-mediated neurodegeneration include blocking the N-methyl-d-aspartate (NMDA)-type glutamate receptor activities, which are closely related to calpain activation; directly inhibiting calpain itself via intrinsic or synthetic calpain inhibitors, or inhibiting its downstream processes; and utilizing the neuroprotectant steroid hormone estrogen and its receptors. In this review, the most remarkable neuroprotective strategies for calpain-mediated neurodegeneration are categorized and summarized with respect to their advantages and disadvantages over one another, in terms of their efficiency and applicability as a therapeutic regimen in the treatment of neurodegenerative diseases.

An updated patent review of calpain inhibitors (2012 - 2014).

INTRODUCTION: Calpain is a family of cysteine proteases found in eukaryotes and a few bacteria. There is considerable interest in the search for calpain inhibitors because the enzyme has been implicated in several diseases including ocular disorders, neurodegenerative disorders, metabolic disorders and cancer. AREAS COVERED: An overview of calpain inhibitors disclosed between 2012 and 2014 is presented. Among these are epoxysuccinates, dipeptide imaging agents, macrocyclic inhibitors, α-helical peptidomimetic inhibitors, carboxamides, 5-azolones and α-mercaptoacrylates. Additionally, preclinical studies of calpain inhibitors in pathologies such blood disorders, ocular disorders, neurological disorders and muscle disorders are discussed. EXPERT OPINION: Major advances made in calpain inhibitor research between 2012 and 2014 include: i) the discovery of cytosolic-stable carboxamide calpain inhibitors; ii) synthesis of epoxysuccinates with excellent bioavailability; iii) disclosure of the X-ray crystal structures of novel α-mercaptoacrylates bound to the pentaEF hand region from human calpain; and iv) disclosure of calpain inhibitors as anti-sickling agents. Several calpain inhibitors were reported but limited effort was directed towards the discovery of calpain isoform selective agents, which continues to dampen the therapeutic potential of calpain inhibitors.

The role of intraplatelet reactive oxygen species in the regulation of platelet glycoprotein Ibα ectodomain shedding.

Glycoprotein (GP) Ibα ectodomain shedding has become a generally accepted negative regulatory mechanism of platelet function. Stimulation of platelet with either physiological or chemical compound results in GPIbα ectodomain shedding in vitro and in vivo, the mechanism, however, is not totally understood. Here we show, collagen, thrombin, and calcium ionophore A23187 induce reactive oxygen species (ROS) generation, and simultaneously incur GPIbα ectodomain shedding. ROS scavengers N-acetylcysteine (NAC) and dithiothreitol (DTT) abolish not only collagen, thrombin, and A23187 induced ROS production, but also GPIbα ectodomain shedding. Interestingly, a recognized calpain activator, dibucaine, induces both ROS production and GPIbα shedding, which are also obviously reduced by NAC and DTT. Furthermore, calpain inhibitors calpain inhibitor I and carbobenzoxy-valinyl-phenylalaninal, obviously reduce dibucaine, thrombin, and A23187-induced ROS generation. These data indicate that ROS plays a key role in collagen, thrombin, and A23187-induced GPIbα ectodomain shedding. Calpain is an up-stream regulator that regulates ROS-mediated GPIbα shedding.