Reactive Centre Loop Mutagenesis of SerpinB3 to Target TMPRSS2 and Furin: Inhibition of SARS-CoV-2 Cell Entry and Replication.

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.

Increased SARS-CoV-2 Infection, Protease, and Inflammatory Responses in Chronic Obstructive Pulmonary Disease Primary Bronchial Epithelial Cells Defined with Single-Cell RNA Sequencing.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation.

Design, synthesis and biological evaluation of 1,5-disubstituted α-amino tetrazole derivatives as non-covalent inflammasome-caspase-1 complex inhibitors with potential application against immune and inflammatory disorders.

Compounds targeting the inflammasome-caspase-1 pathway could be of use for the treatment of inflammation and inflammatory diseases. Previous caspase-1 inhibitors were in great majority covalent inhibitors and failed in clinical trials. Using a mixed modelling, computational screening, synthesis and in vitro testing approach, we identified a novel class of non-covalent caspase-1 non cytotoxic inhibitors which are able to inhibit IL-1ß release in activated macrophages in the low µM range, in line with the best activities observed for the known covalent inhibitors. Our compounds could form the basis of further optimization towards potent drugs for the treatment of inflammation and inflammatory disorders including also dysregulated inflammation in Covid 19.

Vaspin, a novel adipokine in woman granulosa cells physiology and PCOS pathogenesis?

Vaspin is a novel adipokine mainly expressed in visceral adipose tissue and closely related to obesity and insulin-resistance. Currently, data about its ovarian expression are limited to animal models and its role in human reproduction is largely unexplored. Our study's aims were then to characterise vaspin expression in the human ovary and to study in vitro its effects on granulosa cells physiology. Secondly, we assessed vaspin and its receptor GRP78 variations in granulosa cells and follicular fluid of a cohort of 112 infertile women undergoing an in vitro fertilisation procedure and allocated to three groups, each including normal-weight and obese subjects: 34 PCOS patients, 33 women with isolated polycystic ovary morphology (ECHO group) and 45 controls. Vaspin and GRP78 expression in the ovary was assessed by immunohistochemistry, RT-qPCR and Western blot. Granulosa cells and follicular fluid were analysed by RT-qPCR and ELISA, respectively. In vitro, granulosa cells metabolism was studied after stimulation with recombinant human vaspin, with and without a siRNA directed against GRP78. Vaspin was highly expressed in the human ovary and concentration-dependently enhanced granulosa cells steroidogenesis, proliferation and viability through GRP78 (P < 0.0001). Vaspin levels in both granulosa cells and follicular fluid were significantly higher in obese women (P < 0.0001) and in the normal-weight ECHO group (P < 0.001), which also had the highest expression rates of GRP78 (P < 0.05). Although further investigation is needed, vaspin appears as a novel modulator of human granulosa cells physiology and possibly plays a role in PCOS pathogenesis, notably protecting from insulin-resistance induced complications.

Using serpins cysteine protease cross-specificity to possibly trap SARS-CoV-2 Mpro with reactive center loop chimera.

Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin-Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.