Staurosporine-induced cleavage of apoptosis-inducing factor in human fibrosarcoma cells is independent of matrix metalloproteinase-2

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which mediates staurosporine (STS)-induced cell death. AIF cleavage and translocation to the cytosol is thought to be calpain-1-dependent as calpain inhibitors reduced AIF proteolysis. However, many calpain inhibitors also inhibit matrix metalloproteinase-2 (MMP-2) activity, an intracellular and extracellular protease implicated in apoptosis. Here we investigated whether MMP-2 activity is affected in response to STS and if contributes to AIF cleavage. Human fibrosarcoma HT1080 cells were treated with STS (0.1 µM, 0.25-24 hr). A significant increase in cellular MMP-2 activity was seen by gelatin zymography after 6 hr STS treatment, prior to induction of cell necrosis. Western blot showed the time-dependent appearance of two forms of AIF (~60 and 45 kDa) in the cytosol which were significantly increased at 6 hr. Surprisingly, knocking down MMP-2 or inhibiting its activity with MMP-2 preferring inhibitors ARP-100 or ONO-4817, or inhibiting calpain activity with ALLM or PD150606, did not prevent the STS-induced increase in cytosolic AIF. These results show that although STS rapidly increases MMP-2 activity, the cytosolic release of AIF may be independent of the proteolytic activities of MMP-2 or calpain.

The Diverse Calpain Family in Trypanosomatidae: Functional Proteins Devoid of Proteolytic Activity?

Calpains are calcium-dependent cysteine peptidases that were originally described in mammals and, thereafter, their homologues were identified in almost all known living organisms. The deregulated activity of these peptidases is associated with several pathologies and, consequently, huge efforts have been made to identify selective inhibitors. Trypanosomatids, responsible for life-threatening human diseases, possess a large and diverse family of calpain sequences in their genomes. Considering that the current therapy to treat trypanosomatid diseases is limited to a handful of drugs that suffer from unacceptable toxicity, tough administration routes, like parenteral, and increasing treatment failures, a repurposed approach with calpain inhibitors could be a shortcut to successful chemotherapy. However, there is a general lack of knowledge about calpain functions in these parasites and, currently, the proteolytic activity of these proteins is still an open question. Here, we highlight the current research and perspectives on trypanosomatid calpains, overview calpain description in these organisms, and explore the potential of targeting the calpain system as a therapeutic strategy. This review gathers the current knowledge about this fascinating family of peptidases as well as insights into the puzzle: are we unable to measure calpain activity in trypanosomatids, or are the functions of these proteins devoid of proteolytic activity in these parasites?

Cisplatin induced neurotoxicity is mediated by Sarm1 and calpain activation

Cisplatin is a commonly used chemotherapy agent with significant dose-limiting neurotoxicity resulting in peripheral neuropathy. Although it is postulated that formation of DNA-platinum adducts is responsible for both its cytotoxicity in cancer cells and side effects in neurons, downstream mechanisms that lead to distal axonal degeneration are unknown. Here we show that activation of calpains is required for both neurotoxicity and formation of DNA-platinum adduct formation in neurons but not in cancer cells. Furthermore, we show that neurotoxicity of cisplatin requires activation of Sarm1, a key regulator of Wallerian degeneration, as mice lacking the Sarm1 gene do not develop peripheral neuropathy as evaluated by both behavioral or pathological measures. These findings indicate that Sarm1 and/or specific calpain inhibitors could be developed to prevent cisplatin induced peripheral neuropathy.

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

The main protease (Mpro) of SARS-CoV-2 is a key antiviral drug target. While most Mpro inhibitors have a γ-lactam glutamine surrogate at the P1 position, we recently found that several Mpro inhibitors have hydrophobic moieties at the P1 site, including calpain inhibitors II and XII, which are also active against human cathepsin L, a host protease that is important for viral entry. In this study, we solved x-ray crystal structures of Mpro in complex with calpain inhibitors II and XII and three analogs of GC-376. The structure of Mpro with calpain inhibitor II confirmed that the S1 pocket can accommodate a hydrophobic methionine side chain, challenging the idea that a hydrophilic residue is necessary at this position. The structure of calpain inhibitor XII revealed an unexpected, inverted binding pose. Together, the biochemical, computational, structural, and cellular data presented herein provide new directions for the development of dual inhibitors as SARS-CoV-2 antivirals.

Boceprevir, calpain inhibitors II and XII, and GC-376 have broad-spectrum antiviral activity against coronaviruses in cell culture

As the COVID-19 pandemic continues to fold out, the morbidity and mortality are increasing daily. Effective treatment for SARS-CoV-2 is urgently needed. We recently discovered four SARS-CoV-2 main protease (Mpro) inhibitors including boceprevir, calpain inhibitors II and XII and GC-376 with potent antiviral activity against infectious SARS-CoV-2 in cell culture. Despite the weaker enzymatic inhibition of calpain inhibitors II and XII against Mpro compared to GC-376, calpain inhibitors II and XII had more potent cellular antiviral activity. This observation promoted us to hypothesize that the cellular antiviral activity of calpain inhibitors II and XII might also involve the inhibition of cathepsin L in addition to Mpro. To test this hypothesis, we tested calpain inhibitors II and XII in the SARS-CoV-2 pseudovirus neutralization assay in Vero E6 cells and found that both compounds significantly decreased pseudoviral particle entry into cells, indicating their role in inhibiting cathepsin L. The involvement of cathepsin L was further confirmed in the drug time-of-addition experiment. In addition, we found that these four compounds not only inhibit SARS-CoV-2, but also SARS-CoV, MERS-CoV, as well as human coronaviruses (CoVs) 229E, OC43, and NL63. The mechanism of action is through targeting the viral Mpro, which was supported by the thermal shift binding assay and enzymatic FRET assay. We further showed that these four compounds have additive antiviral effect when combined with remdesivir. Altogether, these results suggest that boceprevir, calpain inhibitors II and XII, and GC-376 are not only promising antiviral drug candidates against existing human coronaviruses, but also might work against future emerging CoVs.

TUG is a calpain-10 substrate involved in the translocation of GLUT4 in adipocytes

The calpain-10 (CAPN10) protease is implicated in the translocation of the glucose transporter 4 (GLUT4), which is retained in the Golgi matrix via the Tether containing a UBX domain for GLUT4 (TUG) protein. Insulin stimulation induces the proteolytic processing of TUG, which leads to the translocation of GLUT4 to the cell membrane. We tested whether TUG is a CAPN10 substrate. Proteolysis of TUG by calpains was assessed using a cell-free system containing calpain-1 and TUG. In situ proteolysis of TUG by calpains was demonstrated in 3T3-L1 adipocytes in the presence of insulin or calpain inhibitors to modulate calpain activity. Proteolysis of TUG by CAPN10 was confirmed using transient or stable silencing of CAPN10 in 3T3-L1 adipocytes. Calpains proteolyzed the C-terminus of TUG in vitro. In adipocytes, insulin-induced cleavage of TUG was correlated with the activation of calpains. Treatment with calpain inhibitors reduced TUG cleavage, resulting in impaired GLUT4 translocation without altering Akt phosphorylation. Furthermore, CAPN10 but not calpain-1 or calpain-2 colocalized with GLUT4 in the absence of insulin, and their colocalization was reduced after stimulation with insulin. Finally, we demonstrated that CAPN10 knockdown reduced the proteolysis of TUG without altering the phosphorylation of Akt or the expression of the Usp25m protease. Thus, our results provide evidence that the TUG protein is cleaved by CAPN10 to regulate GLUT4 translocation.

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.