The glycosaminoglycan heparin is known to possess antimetastatic activity in experimental models and preclinical studies, but there is still uncertainty over its mechanism of action in this respect. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III, but a similar cofactor role has not been previously investigated for proteases linked to metastasis. The squamous cell carcinoma antigens (serpins B3 and B4) are tumor-associated proteins that can inhibit papain-like cysteine proteases, including cathepsins L, K, and S. In this study, we show that SCCA-1 (B3) and SCCA-2 (B4) can bind heparin as demonstrated by affinity chromatography, native PAGE gel shifts, and intrinsic fluorescence quenching. Binding was specific for heparin and heparan sulfate but not other glycosaminoglycans. The presence of heparin accelerated inhibition of cathepsin L by both serpins, and in the case of SCCA-1, heparin increased the second order inhibition rate constant from 5.4 x 10(5) to >10(8), indicating a rate enhancement of at least 180-fold. A templating mechanism was shown, consistent with ternary complex formation. Furthermore, SCCA-1 inhibition of cathepsin L-like proteolytic activity secreted from breast and melanoma cancer cell lines was significantly enhanced by heparin. This is the first example of glycosaminoglycan enhancement of B-clade serpin activity and the first report of heparin acting as a cofactor in serpin cross-class inhibition of cysteine proteases. Most importantly, this finding raises the possibility that the anticancer properties of heparin may be due, at least partly, to enhanced inhibition of prometastatic proteases.

PEDF (Pigment epithelium-derived factor) is a non-inhibitory member of the serpin gene family (serpinF1) that displays neurotrophic and anti-angiogenic properties. PEDF contains a secretion signal sequence, but although originally regarded as a secreted extracellular protein, endogenous PEDF is found in the cytoplasm and nucleus of several mammalian cell types. In this study we employed a yeast two-hybrid interaction trap screen to identify transportin-SR2, a member of the importin-β family of nuclear transport karyopherins, as a putative PEDF binding partner. The interaction was supported in vitro by GST-pulldown and co-immunoprecipitation. Following transfection of HEK293 cells with GFP-tagged PEDF the protein was predominantly localised to the nucleus, suggesting that active import of PEDF occurs. A motif (YxxYRVRS) shared by PEDF and the unrelated transportin-SR2 substrate, RNA binding motif protein 4b, was identified and we investigated its potential as a nuclear localization signal (NLS) sequence. Site-directed mutagenesis of this helix A motif in PEDF resulted in a GFP-tagged mutant protein being excluded from the nucleus, and mutation of two arginine residues (R67, R69) was sufficient to abolish nuclear import and PEDF interaction with transportin-SR2. These results suggest a novel NLS and mechanism for serpinF1 nuclear import, which may be critical for anti-angiogenic and neurotrophic function.

The SARS-CoV-2 virus can utilize host cell proteases to facilitate cell entry, whereby the Spike (S) protein is cleaved at two specific sites to enable membrane fusion. Furin, transmembrane protease serine 2 (TMPRSS2), and cathepsin L (CatL) are the major proteases implicated, and are thus targets for anti-viral therapy. The human serpin (serine protease inhibitor) alpha-1 antitrypsin (A1AT) shows inhibitory activity for TMPRSS2, and has previously been found to suppress cell infection with SARS-CoV-2. Here, we have generated modified serpin inhibitors with increased specificity for these cellular proteases. Using SerpinB3 (SCCA-1), a cross-class inhibitor of CatL, as a scaffold, we have designed and produced reactive centre loop (RCL) variants to more specifically target both furin and TMPRSS2. Two further variants were generated by substituting the RCL P7–P1 with the spike protein S1/S2 cleavage site from either SARS-CoV-2 alpha or delta (P681R) sequences. Altered inhibitory specificity of purified recombinant proteins was verified in protease assays, with attenuated CatL inhibition and gain of furin or TMPRSS2 inhibition, as predicted, and modified serpins were shown to block S protein cleavage in vitro. Furthermore, the serpin variants were able to inhibit S-pseudoparticle entry into A549-ACE2-TMPRSS2 cells and suppress SARS-CoV-2 replication in Vero E6 cells expressing TMPRSS2. The construct designed to inhibit TMPRSS2 (B3-TMP) was most potent. It was more effective than A1AT for TMPRSS2 enzyme inhibition (with an eighteen-fold improvement in the second order inhibition rate constant) and for blocking SARS-CoV-2 viral replication. These findings advance the potential for serpin RCL mutagenesis to generate new inhibitors, and may lead to novel anti-viral biological molecules.