The NIH Virtual SARS-CoV-2 Antiviral Summit, held on November 6, 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for COVID-19, including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss SARS-CoV-2 targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

• Genetic deletion of full-length mouse dematin causes severe abnormalities of erythrocyte shape, membrane stability, and hemolytic anemia.• Dematin is critical for the junctional complex integrity.Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase.Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex. (Blood. 2016;128(1):93-103)

Calpain-1, a calcium-activated cysteine protease, is ubiquitously expressed in hematopoietic cells, and is known to play a functional role in a myriad of cellular processes by regulating limited cleavage of multiple substrates.1 Using a calpain-1 null model (CKO) previously generated in our laboratory, recent studies revealed a functional role of calpain-1 in IgE-dependent mast cell activation.2 Interestingly, in the Berkeley sickle mice, mast cell activation contributes to neurogenic inflammation, chronic pain, and hypoxia/reoxygenation (H/R)-evoked hyperalgesia, which were ameliorated upon treatment with mast cell inhibitor imatinib, and cannabinoids as well as nociception receptor ligand AT200. 3-5Therefore, we examined if calpain-1 contributes to chronic and/or hypoxia/reoxygenation (H/R)-evoked acute pain in sickle mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice by cross-breeding HbSS-Townes sickle mice with calpain-1 KO mice. Systemic deletion of calpain-1 in Townes sickle mice 6 ameliorated chronic pain behaviors including mechanical, heat, cold, and deep tissue/musculoskeletal hyperalgesia.Calpains in mammals are encoded by 14 genes; however, two conventional calpains termed calpain-1 (m-calpain) and calpain-2 (m-calpain), with 61% amino acid identity, are highly expressed.1 Calpain-1 is activated at micromolar calcium, whereas active calpain-2 is detected at millimolar calcium concentration in vitro.1 Calpastatin, an endogenous inhibitor of both calpains, provides a regulatory mechanism for suppressing calpain activity under steady state conditions. 1 While both calpains are ubiquitously expressed, calpain-1 generally dominates in hematopoietic cells such as RBCs and platelets. In contrast, calpain-2 is more prominent in the nervous system. Sickle RBCs are known to exhibit high levels of intracellular calcium.7 Our previous study demonstrated that dense/dehydrated RBCs from patients with sickle cell disease show enhanced calpain-1 activity as measured by the calpastatin levels, and pharmacological inhibition of calpain-1 in the transgenic SAD mouse model of mild sickle cell disease reduced sickle RBC density/dehydration. 8Due to high sequence similarity between the two calpains, gene targeting and not the use of pharmacological inhibitors is preferable to elucidate the unique function of each calpain isoform in vivo. While the calpain-1 knockout mice are viable and fertile, 9 calpain-2 gene inactivation causes early embryonic lethality in mice. Following up on our recent investigation of the role of calpain-1 in the SAD mouse model of mild sickle cell disease (SCD), 8in the present study we utilized genetic inactivation of calpain-1 in the homozygous, HbSS-Townes mice with severe SCD to define the role of calpain-1 in pain sensitivity. A comprehensive breeding strategy was designed to generate viable calpain-1 knockout Townes sickle (SSCKO) mice ( Figure 1A and B), which express human but not mouse α-and b-globin genes. After 15 generations of breeding, the SSCKO mice were generated by ...

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5’-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in the hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in the steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa/αIIbβ3 integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD.

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Sickle cell disease (SCD) is complex with disabling and life-threatening consequences including pain and organ damage. We hypothesized that calpain-1, a calcium-dependent protease may contribute to both organ damage and pain in SCD because deletion of calpain-1 in HbSS Townes mice ameliorated hyperalgesia (Nwankwo et al., Haematologica 2016), and BDA-410, a selective calpain-1 inhibitor, significantly improved age-associated organ damage in a mouse model of aging (Nabeshima et al., Sci Reports 2014). We performed a randomized double-blind placebo-controlled trial in homozygous female HbSS BERK (sickle) mice to examine the effect of BDA-410 on pain and organ pathology. Mice were treated daily intraperitoneally with vehicle (50 μl DMSO in 950 μl sesame oil) and BDA-410 at 30 mg/kg or 100 mg/kg for 2 weeks. Mechanical-, thermal- (heat and cold), and deep hyperalgesia were assessed at baseline (before treatment), during treatment (1-hour post-injection, as well as on days 4, 8, 12), and after discontinuation of treatment (days 16, 20, 24, and 28). Using 2-way repeated measures ANOVA with Tukey's correction, we observed a significant decrease in mechanical, heat, and cold hyperalgesia in sickle mice treated with BDA-410. Compared to baseline, we observed a significant decrease in, [a] mechanical hyperalgesia, on day 8 with 30 mg/kg (p=0.0312), and days 8, 12, 16, 20, and 24 with 100 mg/kg dose (p=0.0012; p<0.0001; p<0.001; p=0.0006 and p=0.0007, respectively); [b] heat hyperalgesia, on days 8, 12, 16, and 20 with 30 mg/kg (p=0.044, 0.0027, 0.0172 and 0.0326, respectively), and on day 12 with 100 mg/kg (p=0.0138); and [c] cold hyperalgesia on days 8, 12, 16, 20, 24, and 28 with both 30 mg/kg (p= 0.0254, 0.0155, 0.001, 0.003, 0.035 and 0.008, respectively) and 100 mg/kg (p= 0.0054, 0.0035, 0.005, 0.009, 0.011 and 0.003, respectively). No significant difference in deep hyperalgesia was observed with either treatment. No significant effect of vehicle was found on any measures. Although BDA-410 treatment was discontinued after 2 weeks, analgesic effect was maintained for a significantly longer time, suggesting amelioration of the pathobiology of pain and/or inhibition of neural transmission of pain mechanisms. Therefore, BDA-410 is effective in reducing chronic pain in sickle mice without causing tolerance. In addition to pain, extensive Purkinje cell damage in the cerebellum and liver infarcts have been demonstrated in BERK sickle mice (Manci et al., Blood 2006). We observed reduced liver infarction in number and area as well as a significant decrease in Purkinje cell damage in sickle mice treated with 30 mg/kg and 100 mg/kg BDA-410 vs. vehicle (p= 0.0007 & 0.0233, respectively). Thus, BDA-410 is effective at significantly reducing Purkinje cell damage in the brain, which is associated with motor dysfunction as well as pain. We therefore examined gait parameters using Mouse Walker equipment which captures natural walking gait based on optical total internal reflection (TIR). Foot contacts disrupt the effect causing frustrated total internal reflection (fTIR) resulting in illuminated points of contact detected by a high-speed camera (Lumenera Lt425C) and a high-performance recording software (StreamPix 7, Norpix). Gait-related parameters were extracted using MouseWalker software. Sickle mice showed significantly lower walking speed (p=0.0062), increased stance duration and stance instability (p= 0.001 and 0.030, respectively) compared to control mice. Gait parameters correlated positively for mechanical hyperalgesia with stance duration (r=0.72, p=0.004) and negatively for grip force with stance instability and stance duration (r = -0.70, p=0.0075 and r = -0.84, p=0.0002). Since decreased grip force demonstrates increased hyperalgesia, deep hyperalgesia also correlates positively with stance instability and stance duration. Thus, Purkinje cell damage is associated with alterations in gait and pain in sickle mice. Since BDA-410 inhibited Purkinje cell damage and reduced pain, it demonstrates the potential of targeting calpain-1 as a treatable target to develop novel therapeutics for treating pain and organ damage in SCD. These data also suggest that gait measures may serve as biomarkers for Purkinje cell damage and pain. We speculate that gait measures may have utility as a diagnostic and prognostic tool in the progression of SCD. BDA-410 and funding in part were provided by 1910 Genetics. Disclosures Pagare: 1910 Genetics: Employment. Nwankwo:1910 Genetics: Equity Ownership. Gupta:1910 Genetics: Research Funding.

Introduction: With an estimated 15 million patients and no drug that addresses its etiology, sickle cell disease (SCD) remains an area of unmet need. Vaso-occlusive pain crisis (VOPC), the hallmark of SCD, is initiated by sickle RBCs (sRBCs) recruiting leukocytes and platelets to potentiate vessel occlusion. ADP released by sRBCs is a potent activator of platelets, and sickle cell patients are known to have activated platelets in circulation both at steady state and during VOPC. However, the mechanism underlying platelet dysfunction in SCD is not fully understood. Platelet activation mediated by the protease activated receptors (PAR1 and PAR4 in humans, PAR3 and PAR4 in mice), triggers PLC-β activation resulting in calcium mobilization. The increased calcium flux leads to activation of GPIIbIIIa/aIIbb3, GP1b, and P-selectin involved in platelet aggregation, adhesion, and rolling. Prior evidence has established a role of the calcium-activated cysteine protease, calpain-1 in platelet activation. Washed platelets from calpain-1 knockout C57BL/6 mice demonstrated impaired platelet aggregation. However, due to the critical contribution of sRBCs to platelet dysfunction in SCD, whole blood (impedance) aggregometry represents a physiological assessment of platelet aggregation. Methods: Townes SCD mice (SS) were backcrossed with calpain-1 knockout (CKO) mice to generate SCD mice lacking calpain-1 (SSCKO). Humanized mice (AA) were used as controls. Using flow cytometry, we evaluated in vivo platelet activation following stimulation with ADP and Thrombin GPRP. Platelet counts were obtained via ADVIA 120 and flow cytometry. For platelet aggregation, 500 μL of blood was harvested from the vena cava of AA, SS, SSCKO, and CKO mice. Whole blood aggregation in response to PAR4 stimulation was assessed using the Roche Multiplate Analyzer. A separate group of mice were challenged with hypoxia/reoxygenation (H/R) treatment (3 hours of 10.5% O2, followed by 4 hours of 21% O2) prior to platelet aggregation testing. SCD mice are characterized by tissue infarcts suggestive of thrombus formation. To examine whether H/R treatment induces formation of fibrin thrombi, we harvested brain, lungs, heart, kidneys, liver, and spleen following blood collection, and performed histology. Results: Compared to AA, SS and SSCKO mice are thrombocytopenic. Similar to Berkeley, Townes mouse platelets are activated in vivo, demonstrated by activated GPIIbIIIa on circulating platelets. At steady state, PAR4 agonist-induced platelet aggregation is similar in AA and SS mice (64 U v. 53 U, n = 6-10/group, p = 0.3). As depicted in Fig 1., SSCKO mice show significantly reduced platelet aggregation compared to SS mice (13 U v 53 U, p<0.001, n = 6-10 per group). As expected, H/R treatment induces platelet hyperactivity in SS, but not AA mice (71 U v. 59 U, p = 0.04, n = 6/group). Interestingly, the H/R-induced platelet hyperactivity partially relieves the calpain-1-mediated inhibition of platelet aggregation in SSCKO mice at steady state (Fig 2) (53 U v. 13 U, p<0.001, n = 6-8/group). Furthermore, CKO mice do not show defective platelet aggregation at both steady state, and following H/R treatment, indicating an important role for sRBCs and adhesion in whole blood aggregation. Despite attenuated platelet aggregation, SSCKO platelets at steady state show comparable expression of activated GPIIbIIIa relative to their AA and SS counterparts, indicating that the impaired aggregation at steady state is not due to defective integrin translocation. Since the Roche Multiplate Analyzer takes platelet adhesion into account in determining aggregation, we hypothesize that defective SSCKO whole blood aggregation at steady state is due to reduced cell adhesion. Finally, while silent infarcts were detected in all tissues, fibrin thrombi were not detected, a finding consistent with studies in the Berkeley model reporting minimal thrombi formation in tissues. In summary, we report that calpain-1 is required for platelet aggregation in steady state SCD but not following H/R treatment. This is the first study of H/R-induced platelet dysfunction in the Townes model, and raises the possibility of targeting calpain-1 as a treatment for platelet hyperactivity in SCD. Figure 1. Impaired steady state whole blood platelet aggregation in SSCKO mice (panel 3). Figure 1. Impaired steady state whole blood platelet aggregation in SSCKO mice (panel 3). Figure 2. H/R relieves calpain-1-mediated inhibition of platelet aggregation in SS-CKO mice (panel 3). Figure 2. H/R relieves calpain-1-mediated inhibition of platelet aggregation in SS-CKO mice (panel 3). Disclosures Jakubowski: Eli Lilly and Company: Employment, Equity Ownership. Frelinger:GL Synthesis: Research Funding; Celerion: Research Funding; Megakaryon: Research Funding; Bristol-Myers Squibb: Research Funding; Sysmex: Research Funding; Eisai: Research Funding; Baxalta: Research Funding; Pfizer: Research Funding; GE Global Research: Research Funding; NIH: Research Funding.

Introduction: Sickle cell disease (SCD) is caused by the substitution of a valine residue for glutamic acid in the β-globin chain of hemoglobin. The resulting sickle hemoglobin rapidly polymerizes upon deoxygenation, forming sickle RBCs (sRBCs) with reduced deformability. Less deformable sRBCs have an impaired ability to navigate the microvasculature, thereby potentiating vaso-occlusive crises, the hallmark of SCD. Deoxygenation-mediated sickling also induces influx of calcium leading to calpain-1 mediated RBC dehydration via the Gardos channel. Calpains are calcium-activated cysteine proteases involved in numerous cellular processes. In mammals, calpain-1 and calpain-2 are the dominantly expressed isoforms, along with the endogenous inhibitor, calpastatin. Previously, we have shown that genetic deletion of calpain-1 improves RBC deformability in C57BL/6 mice (Wieschhaus et al, Biochem J 2012). Similarly, pharmacological inhibition of calpain-1 prevents dehydration of sRBCs in the SAD model of mild SCD (De Franceschi, et al, FASEB J 2013). Here, our objective is to determine whether genetic deletion of calpain-1 in the HbSS-Townes mice – a severe SCD mouse model, improves RBC deformability in both normoxic and hypoxic conditions. Methods: Transgenic SCD mice (HbSS-Townes) expressing >99% human HbS were backcrossed with calpain-1 knockout (CKO) mice to obtain sickle mice lacking calpain-1 (HbSS-Townes-CKO). Casein zymography was performed to confirm the complete loss of calpain-1 activity in the RBCs of the HbSS-Townes-CKO mice. Thiazole orange-based FACS revealed that HbSS-Townes and HbSS-Townes-CKO mice express ~60% reticulocytes. The elevated reticulocyte count prompted us to evaluate the effect of calpain-1 deficiency on reticulocyte deformability. We utilized a microfluidic-based flow cytometer to assess single reticulocyte deformability following thiazole orange staining upon treatment with calcium ionophore (A23187) and CaCl2. We further probed the molecular mechanisms of RBC deformability by assessing calcium-induced proteolysis of RBC cytoskeletal proteins. RBCs were harvested from HbSS-Townes and HbSS-Townes-CKO mice, treated with A23187 and CaCl2 to activate calpain-1, and subjected to RBC ghosts preparation and gel electrophoresis. Cytoskeletal proteins were visualized and quantified. Guided by our previous findings that calpain-1 knockout (CKO) mice exhibit a clot retraction defect (Kuchay, et al, MCB 2007), we harvested un-coagulated blood from HbSS-Townes and HbSS-Townes-CKO mice, and assessed their clot formation phenotype in calcium-free RPMI medium at different time intervals. Results: Casein zymography revealed a complete loss of calpain-1 enzyme activity (indicated by lack of casein cleavage) in the HbSS-Townes-CKO mice. Furthermore, reticulocytes from HbSS-Townes-CKO mice are more deformable than their HbSS-Townes counterparts under steady state as well as upon treatment with calcium ionophore and CaCl2. An improvement in sickle reticulocyte deformability has significant potential to prevent vaso-occlusive crisis under situations of hypoxia-induced ischemia reperfusion injury – a hypothesis that is currently being tested. In addition, our preliminary results show that RBC ghosts from HbSS-Townes mice exhibit a transglutaminase-mediated cross-linking defect in the cytoskeleton that is restored in the HbSS-Townes-CKO mice. This finding has significant implications for an increased in vivo RBC life span of HbSS-Townes-CKO mice. Finally, we found that HbSS-Townes-CKO mice display a whole blood clot retraction defect, similar to our previous findings on platelets harvested from calpain-1 knockout (CKO) mice. Our findings showing improved sickle cell deformability and clot retraction phenotype are consistent with recently published findings demonstrating an interaction between RBCs and platelets in clot formation and in vivo thrombosis. To test this intriguing hypothesis, we are currently evaluating whether HbSS-Townes-CKO mice exhibit reduced thrombus formation upon laser-induced in vivo thrombosis. Together, our results report the first mouse model of severe sickle cell disease lacking calpain-1 activity, and unveil a new role for calpain-1 in the multi-step process regulating SCD pathogenesis. These findings might serve as a basis for exploring calpain-1 inhibition as a therapeutic target for SCD. Disclosures No relevant conflicts of interest to declare.