Glycoprotein Inhibitors Research Articles

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

Dynamics of hemostasis system parameters in assessing the risk of complications in the patients with acute myocardial infarction receiving antiplatelet therapy

Background. Current treatment of patients with myocardial infarction is based on the strategy of early invasive coronary intervention in combination with dual antiplatelet therapy - with acetylsalicylic acid and a P2Y12 blocker of platelet adenosine diphosphate receptors. In patients with thrombosis of the infarct-related artery, the phenomenon of “slow/ no reflow” (slowing of blood flow due to distal embolization of the artery), inhibitors of glycoprotein IIb/IIIa platelet receptors are administered as additional disaggregant therapy. In patients undergoing standard antiplatelet therapy in combination with glycoprotein IIb/IIIa inhibitors, there is a risk of hemorrhagic complications, therefore, monitoring of hemostasis parameters is necessary. Currently, there are no standard approaches to monitor the antiplatelet therapy.Objective. To study the dynamics of hemostatic system parameters in patients with acute myocardial infarction during antiplatelet therapy.Material and methods. We assessed platelet aggregation with 10 µmol of adenosine phosphate as an inducer in patients with ST-segment elevation myocardial infarction with different options of standard antiplatelet therapy in combination with GPIIb/IIIa inhibitors. Group 1 included 20 patients on dual antiplatelet therapy (clopidogrel 75 mg + acetylsalicylic acid 100 mg) + GPIIb/IIIa inhibitor (tirofiban). Group 2 included 15 patients on dual antiplatelet therapy (ticagrelor 180 mg + acetylsalicylic acid 100 mg) + GPIIb/IIIa inhibitor.Results. While on antiplatelet therapy the patients in both groups 1 and 2 demonstrated a decrease in platelet aggregation ability under the impact of adenosine phosphate, relative to the norm: 38 (21;43) % and 14 (11;15) %, respectively, the norm being 79 (73;84) % (p<0.05). Meantime, no thrombotic events in the form of stent thrombosis were noted, which indicated antiplatelet therapy efficacy. In an intragroup comparison, the decrease in the platelet aggregation ability was most pronounced in group 2 (p<0.05). By the 7th day of myocardial infarction, the platelet aggregation had continued to decrease to 26 (17;43) % in group 1, to 10 (7;11) % in group 2. The most pronounced effect of antiplatelet therapy was observed in group 2 (p<0.05), which was manifested by a statistically significant decrease in platelet aggregation ability under the impact of 10 µmol of adenosine phosphate.Conclusions. While studying the hemostasis system changes over time in patients with myocardial infarction receiving antiplatelet therapy, we have found that making the platelet aggregation ability assessment with 10 µmol of adenosine phosphate as an inducer is possible to identify the effect of medications. However, further studies including larger patient groups are needed to determine the target values of platelet aggregation with 10 µmol of adenosine phosphate and assess the therapy efficacy.

Incidence of intrahospitalary major cardiac events with the use of mechanic thromboaspiration versus only use of IIB/IIIA glucoprotein inhibitors on patients with acute ST elevation myocardial infarction

To identify the incidence of in-hospital major adverse cardiac events (MACE) with the use of mechanic thromboaspiration plus IIb/IIIa glycoprotein inhibitors versus only use of IIb/IIIa glycoprotein inhibitors on patients with acute ST elevation myocardial infarction. Retrospective, observational, cohort analytic study, on patients with acute ST elevation myocardial infarction that had angiography thrombus TIMI 5 grade, treated between October 2021 and December 2022. A total of 237 patients were included. In 113 patients thromboaspiration were used, 124 patients didn't used. 81.6% were men. In-hospital MACE occurred on 31.9% of patients with thromboaspiration use vs. 30.6% on patients with no use (RR: 1.05; IC95%: 0.61-1.93; p = 0.840). Incidence of malignant arrhythmias were of 8% with thromboaspiration use vs. 1.6% on patients with no use (RR: 5.27; IC95%: 1.11-24.97; p = 0.020). The use of thromboaspiration on concomitant treatment with IIb/IIIa glycoprotein inhibitors was similar with only IIb/IIIa glycoprotein inhibitors in reducing incidence of in-hospital MACE on patients with ST elevation acute myocardial infarction and high thrombus burden. The study has several limitations, so results should be taken with caution.

Effect of drug interactions with non-vitamin-K oral anticoagulants on thromboembolic events in patients with nonvalvular atrial fibrillation.

Few real-world studies have investigated drug-drug interactions (DDIs) involving non-vitamin-K antagonist oral anticoagulants (NOACs) in patients with nonvalvular atrial fibrillation (NVAF). The interactions encompass drugs inducing or inhibiting cytochrome P450 3A4 and permeability glycoprotein. These agents potentially modulate the breakdown and elimination of NOACs. This study investigated the impact of DDIs on thromboembolism in this clinical scenario. Patients who had NVAF and were treated with NOACs were selected as the study cohort from the National Health Insurance Research Database of Taiwan. Cases were defined as patients hospitalised for a thromboembolic event and who underwent a relevant imaging study within 7 days before hospitalisa-tion or during hospitalisation. Each case was matched with up to 4 controls by using the incidence density sampling method. The concurrent use of a cytochrome P450 3A4/permeability glycoprotein inducer or inhibitor or both with NOACs was identified. The effects of these interactions on the risk of thromboembolic events were examined with univariate and multivariate conditional logistic regressions. The study cohort comprised 60,726 eligible patients. Among them, 1288 patients with a thromboembolic event and 5144 matched control patients were selected for analysis. The concurrent use of a cytochrome P450 3A4/permeability glycoprotein inducer resulted in a higher risk of thromboembolic events (adjusted odds ratio [AOR] 1.23, 95% confidence interval [CI] 1.004-1.51). For patients with NVAF receiving NOACs, the concurrent use of cytochrome P450 3A4/ permeability glycoprotein inducers increases the risk of thromboembolic events.

Glycoprotein inhibitors as a first line rescue treatment after unsuccessful recanalization of endovascular thrombectomy: A systematic review and meta-analysis.

Intracranial atherosclerotic disease (ICAD) is a major cause of stroke with a high rate of re-occlusion following mechanical thrombectomy (MT). Among the available rescue options, glycoprotein IIb/IIIa inhibitors (GPI) have shown promise as a potential therapeutic strategy. This systematic review and meta-analysis examine studies exploring the use of glycoprotein inhibitors as a first-line treatment for refractory occlusion or high-grade stenosis following EVT in the setting of ICAD. A systematic review and meta-analysis were performed. Studies using GPI as the first-line rescue treatment (GPI-rt) after failed thrombectomy or in the setting with high-grade stenosis (>50%) were included. The primary outcome of interest was good clinical outcomes (defined as a modified Rankin Scale (mRS) score of 0-2 at 90 days). Secondary outcomes of interest were successful recanalization (TICI 2b-3), symptomatic intracranial hemorrhage (sICH), and mortality by 90 days. Our study processed 2111 articles, which yielded eight relevant studies for review, four single and four double arm. These studies comprised 763 patients, divided into GPI-rt (535 patients) and non-GPI-rt (228 patients) cohorts. The GPI-rt group had higher rates of mRS ≤ 2 at 90 days (58.5% vs 38.9%, p = 0.002) and lower mortality rates (7.8% vs 17.5%, p = 0.04) compared to the non-GPI-rt cohort. mTICI 2b-3 rates and rates of sICH were not significantly different between the cohorts. First line GPI-rt demonstrates significant clinical benefit and significantly lower mortality without a rise in rates of sICH. GPI are a potential first line rescue treatment of ICAD.

Critical Analysis of Thrombocytopenia Associated With Glycoprotein IIb/IIIa Inhibitors and Potential Role of Zalunfiban, a Novel Small Molecule Glycoprotein Inhibitor, in Understanding the Mechanism(s).

Thrombocytopenia is a rare but serious complication of the intravenous glycoprotein IIb/IIIa (GPIIb/IIIa; integrin αIIbβ3) receptor inhibitors (GPIs), abciximab, eptifibatide, and tirofiban. The thrombocytopenia ranges from mild (50 000-100 000 platelets/μL), to severe (20 000 to <50 000/μL), to profound (<20 000/μL). Profound thrombocytopenia appears to occur in <1% of patients receiving their first course of therapy. Thrombocytopenia can be either acute (<24 hours) or delayed (up to ~14 days). Both hemorrhagic and thrombotic complications have been reported in association with thrombocytopenia. Diagnosis requires exclusion of pseudothrombocytopenia and heparin-induced thrombocytopenia. Therapy based on the severity of thrombocytopenia and symptoms may include drug withdrawals and treatment with steroids, intravenous IgG, and platelet transfusions. Abciximab-associated thrombocytopenia is most common and due to either preformed antibodies or antibodies induced in response to abciximab (delayed). Readministration of abciximab is associated with increased risk of thrombocytopenia. Evidence also supports an immune basis for thrombocytopenia associated with the 2 small molecule GPIs. The latter bind αIIbβ3 like the natural ligands and thus induce the receptor to undergo major conformational changes that potentially create neoepitopes. Thrombocytopenia associated with these drugs is also immune-mediated, with antibodies recognizing the αIIbβ3 receptor only in the presence of the drug. It is unclear whether the antibody binding depends on the conformational change and whether the drug contributes directly to the epitope. Zalunfiban, a second-generation subcutaneous small molecule GPI, does not induce the conformational changes; therefore, data from studies of zalunfiban will provide information on the contribution of the conformational changes to the development of GPI-associated thrombocytopenia.

Acute Respiratory Distress Syndrome: A Case Report and Review on Diagnosis and Novel Interventions in Management

Acute respiratory distress syndrome is a type of acute diffuse lung injury, frequently brought on by sepsis, trauma, and severe pulmonary infections and continues to be one of the major causes of morbidity and mortality in critically ill patients, despite the constantly involving advancements. The management strategies have constantly evolved with a wide array of ventilation strategies, conservative fluid management, neuromuscular blockade. As we move into the post pandemic Era, the mortality rate due to ARDS continues to remain high hence improving patient outcomes is the primary goal of the various treatment approaches. The two most effective techniques are still prone ventilation and lung protection ventilation. In this report we aim to present the case of a 30-year old female who has undergone left lobectomy in childhood and presents to the clinic with palpitations, fever, shortness of breath and spasmodic cough with phlegm. The workup included chest Xray, CT scan, arterial blood gas with electrolytes, echocardiography and urinanalysis. Based on clinical and imaging findings, the diagnosis of acute respiratory distress syndrome was made. Against the background of intensive treatment, the patient’s condition improved to some extent, with positive dynamics. Lung protective ventilation, prone ventilation, high frequency oscillatory ventilation among many other novel interventions and futuristic direction of ARDS with mesenchymal stromal cells and Ulinastatin, a urinary glycoprotein and protease inhibitor provides a ray of hope in the management of this acute condition. Keywords: Acute respiratory distress syndrome; hypoplasia; lung ventilation; prone ventilation; mesenchymal stem cells; ulinastatin

N-linked glycoproteins and host proteases are involved in swine acute diarrhea syndrome coronavirus entry.

Gaining insight into the cell-entry mechanisms of swine acute diarrhea syndrome coronavirus (SADS-CoV) is critical for investigating potential cross-species infections. Here, we demonstrated that pretreatment of host cells with tunicamycin decreased SADS-CoV attachment efficiency, indicating that N-linked glycosylation of host cells was involved in SADS-CoV entry. Common N-linked sugars Neu5Gc and Neu5Ac did not interact with the SADS-CoV S1 protein, suggesting that these molecules were not involved in SADS-CoV entry. Additionally, various host proteases participated in SADS-CoV entry into diverse cells with different efficiencies. Our findings suggested that SADS-CoV may exploit multiple pathways to enter cells, providing insights into intervention strategies targeting the cell entry of this virus.

A Review of Antithrombotic Agents for the Treatment of Ischemic Stroke

Ischemic stroke caused by a blockage in blood vessels that supplies blood to the brain, can lead to severe neurological disability or even death if not treated in a timely proper manner. Current treatment for ischemic stroke comprises Intravenous Thrombolytic Therapy (IVT) and Endovascular Therapy (EVT). However, the unmet medical needs lie in the high disability, narrow therapeutic window, low patient eligibility for operations, and recurrent ischemic stroke and bleeding complications. The development of antithrombotic agents is key to reducing complications of current treatment and secondary prevention of ischemic stroke. This review paper covers a wide range of most commonly used antithrombotic medications or advancements in antithrombotic agents, including warfarin, heparins (UFH and LMWH), direct oral anticoagulants (dabigatran, rivaroxaban, apixaban), aspirin, dual-antiplatelet therapies (clopidogrel/aspirin and dipyridamole/aspirin), and Glycoprotein (GP) inhibitors (tirofiban and anfibatide). This paper compares the efficacy and safety of antithrombotic agents and discusses the advancement and potential of GP inhibitors in treating ischemic stroke patients. Based on preclinical and clinical results, it was found that GP inhibitors have higher antithrombotic and neuroprotective efficacy and are relatively safe with a lower rate of bleeding complications among all agents. The novel Anfibatide, a snake venom-derived GPIba inhibitor currently in the early clinical development stage, has shown superior efficacy and safety profile compared to other agents. It has great potential to fulfill the current unmet medical needs in ischemic stroke treatment. It is worth exploring the mechanism of GP inhibitors and their applications in ischemic stroke treatment.

Abstract P104: EXAMINING THE EFFECTS OF FOLLISTATIN ON CONTRACTION OF VESSELS FROM NORMOTENSIVE AND HYPERTENSIVE RATS

The lab has previously shown that follistatin (FST), a glycoprotein inhibitor of activins, reduced blood pressure in the Spontaneously Hypertensive Rat (SHR) model of Essential Hypertension. Short-term exposure (30 minutes) to FST also reduced vessel contraction induced by potassium (KCl). This study aims to illustrate the mechanisms through which FST inhibits potassium-induced contraction in SHR and normotensive (WKY) rats. Function of SHR or WKY resistance mesenteric arteries was tested in response to KCl and ion channel modulators using wire myography. Cultures of primary smooth muscle cells (SMC) were also established from these vessels using cell explant technique. The calcium sensor dye Fluo-4 AM was used to examine calcium influx on a cellular level. To test whether FST effects were due to activin neutralization or activin-receptor inhibition, neutralizing antibodies against activin A and B or an activin receptor inhibitor (SB 431542) were used, respectively. Only activin A neutralization in the SHR reduced KCl-induced contraction similarly to FST. Activin receptor inhibition also reduced KCl constriction similarly to FST. When directly stimulated with Activin A, both WKY and SHR vessels displayed augmented KCl contraction compared to controls but the effect was more pronounced in the SHR. Preliminary Fluo-4 results showed that FST decreased KCl-induced calcium influx in SMC. Since activin A was shown to increase L-type voltage-gated Ca2+channel activity, an agonist (FPL 64176) of this channel was used to induce contraction. Although FST reduced KCl-induced contraction, it did not affect FPL-induced contraction in either SHR or WKY. Thus, Activin A neutralization reproduces FST reduction of KCl-induced vessel contraction. FST did not reduce FPL-induced contraction, suggesting that its inhibition of KCl-induced contraction is not through effects on this channel but rather an alternate Ca2+ or K-ATP channel.

De novo design of bioactive phenol and chromone derivatives for inhibitors of Spike glycoprotein of SARS-CoV-2 in silico.

This work presents the synthesis of 12 phenol and chromone derivatives, prepared by the analogs, and the possibility of conducting an in silico study of its derivatives as a therapeutic alternative to combat the SARS-CoV-2, pathogen responsible for COVID-19 pandemic, using its S-glycoprotein as a macromolecular target. After the initial screening for the ranking of the products, it was chosen which structure presented the best energy bond with the target. As a result, derivative 4 was submitted to a molecular growth study using artificial intelligence, where 8436 initial structures were obtained that passed through the interaction filters and similarity to the active glycoprotein pocket through the MolAICal computational package. Thus, 557 Hits with active configuration were generated, which is very promising compared to the BLA reference link for inhibiting the biological target. Molecular dynamics also simulated these compounds to verify their stability within the active protein site to seek new therapeutic propositions to fight against the pandemic. The Hit 48 and 250 are the most active compounds against SARS-CoV-2. In summary, the results show that the Hit 250 would be more active than the natural compound, which could be further developed for further testing against SARS-CoV-2. The study employs the de novo approach to design new drugs, combining artificial intelligence and molecular dynamics simulations to create efficient molecular structures. This research aims to contribute to the development of effective therapeutic strategies against the pandemic.

4'-(8-(4-Methylimidazole)-octyloxy)-arctigenin: The first inhibitor of fish rhabdovirus glycoprotein.

Spring viraemia of carp virus (SVCV), a highly pathogenic rhabdovirus, could cause spring viraemia of carp (SVC) with up to 90% lethality. Like other rhabdoviruses, the entry of SVCV into susceptible cells was mediated by a single envelope glycoprotein G. Specific inhibitors targeting the glycoprotein were the most effective means to alleviate the epidemic. The programs including SWISS-MODEL, I-TASSER, Phyre2 and AlphaFold2 were used to build a three-dimensional structural model of glycoprotein. The structural comparison between SVCV-G and homology protein VSV-G revealed that the SVCV glycoprotein ectodomain (residues 19 to 466) folded into four distinct domains. Based on the potential small molecule binding sites on glycoprotein surfaces, virtual screening of the anti-SVCV drug libraries was performed using Autodock software and 4'-(8-(4-Methylimidazole)-octyloxy)-arctigenin (MOA) with a high binding affinity was identified. The solubility enhancer tags including trigger factor and maltose binding protein were fused with the ectodomain of glycoprotein, and the target protein with a purity of about 90% was successfully obtained. The interaction confirmation tests revealed that the fluorescence intensity of a characteristic peak induced by the endogenous chromophores in glycoprotein was decreased with the addition of MOA, indicating changes in the microenvironment of glycoprotein. Moreover, the interaction could cause a slight conformational change in glycoprotein, as shown by the content of β-turn, β-folding, and random coil of protein all increased with the decrease of α-helix content after the addition of MOA compound. These results demonstrated that MOA could act as a novel drug against fish rhabdovirus via direct targeting of glycoprotein.

Evaluation of the interaction between potent small molecules against the Nipah virus Glycoprotein in Malaysia and Bangladesh strains, accompanied by the human Ephrin-B2 and Ephrin-B3 receptors; a simulation approach.

Malaysia reported the first human case of Nipah virus (NiV) in late September 1998 with encephalitis and respiratory symptoms. As a result of viral genomic mutations, two main strains (NiV-Malaysia and NiV-Bangladesh) have spread around the world. There are no licensed molecular therapeutics available for this biosafety level 4 pathogen. NiV attachment glycoprotein plays a critical role in viral transmission through its human receptors (Ephrin-B2 and Ephrin-B3), so identifying small molecules that can be repurposed to inhibit them is crucial to developing anti-NiV drugs. Consequently, in this study annealing simulations, pharmacophore modeling, molecular docking, and molecular dynamics were used to evaluate seven potential drugs (Pemirolast, Nitrofurantoin, Isoniazid Pyruvate, Eriodictyol, Cepharanthine, Ergoloid, and Hypericin) against NiV-G, Ephrin-B2, and Ephrin-B3 receptors. Based on the annealing analysis, Pemirolast for efnb2 protein and Isoniazid Pyruvate for efnb3 receptor were repurposed as the most promising small molecule candidates. Furthermore, Hypericin and Cepharanthine, with notable interaction values, are the top Glycoprotein inhibitors in Malaysia and Bangladesh strains, respectively. In addition, docking calculations revealed that their binding affinity scores are related to efnb2-pem (- 7.1kcal/mol), efnb3-iso (- 5.8kcal/mol), gm-hyp (- 9.6kcal/mol), gb-ceph (- 9.2kcal/mol). Finally, our computational research minimizes the time-consuming aspects and provides options for dealing with any new variants of Nipah virus that might emerge in the future.

Abstract TP222: Aging-associated Medin Induces Human Brain Microvascular Endothelial Cell Pro-inflammatory And Pro-thrombotic Activation Partly Via NFκB

Background: Vascular aging is characterized by endothelial dysfunction and proinflammatory &amp; prothrombotic changes. It is responsible for aging-associated pathologies including stroke, vascular dementia (VaD) and Alzheimer’s disease (AD). The underlying mechanisms of vascular aging are poorly understood. Medin is an amyloidogenic protein that accumulates in aging vasculature, is associated with VaD and AD, and is a candidate mediator of vascular aging through unknown mechanisms. Our aim is to test the hypothesis that medin induces proinflammatory and prothrombotic changes in human brain microvascular endothelial cells (HBMVECs) through nuclear factor kappa B (NFκB). Methods: HBMVECs were exposed to vehicle, recombinant medin (5 μM, a physiologically relevant dose) without or with RO106-9920 (a small molecule selective NFκB inhibitor, 10 μM) or RO106-9920 alone for 20 hours (N=3-17/treatment). Cell lysates were measured for gene expression of inflammatory cytokines/chemokines (IL-8, IL-6, ICAM-1 and VCAM-1) and thrombotic factors (thrombomodulin (TM), a transmembrane glycoprotein and potent inhibitor of coagulation, plasminogen activator inhibitor-1 (PAI-1) which promotes thrombosis and tissue factor (TF) which initiates the coagulation cascade), using real time PCR. Results: See Figure. Medin increased gene expression of IL-8, IL-6, ICAM-1, VCAM-1 and PAI-1, reduced TM with no change in TF. Co-treatment of medin with RO106-9920 attenuated the increases in IL-8, IL-6, ICAM-1, VCAM-1 and the reduction in TM, with no change in PAI-1. Conclusions: Medin caused increased pro-inflammatory gene expression in HBMVECs. It also caused reduced TM and increased PAI-1 gene expression, signifying prothrombotic activation. Co-treatment with NFκB inhibitor prevented medin’s proinflammatory effect, and effect on TM, but not PAI-1. The effect of inducing endothelial cell activation supports medin as a potential novel target to treat vascular aging.

Virtual Screening on Indonesian Herbal Compounds as SARS-CoV-2 Spike (S2) Glycoprotein Inhibitors: Pharmacophore Modelling &amp; Molecular Docking Approaches

Background: There are still no specific treatments for coronavirus disease (COVID-19) until present days. Several studies have been conducted to determine whether herbal medicine can be an option to be used as a definitive medicine for COVID-19. S2 subunit of spike protein which is responsible for SARS-CoV-2 entry to the host cell, is a potential drug target to inhibit the viral infection. In this study, we aimed to find some compounds from the HerbalDB database that have potential as SARS-CoV-2 spike (S2) glycoprotein inhibitor. Methods: The 6LXT protein was used as the target protein. The procedure in this study consisted of these following steps: protein and ligand preparation, pharmacophore modelling and compound screening, molecular docking, ADME, and toxicity analysis. The docking of hit compounds to the target protein were compared to arbidol and quercetin as positive controls. Results: Four hit compounds were screened from HerbalDB compounds. Two of them, octopamine and L-noradrenaline, showed lower binding energies (respectively, -5.19 and -4.98 kcal/mol) than positive controls whereas the other two compounds, mimosine and L-theanine, showed higher binding energies (respectively, -3.99 and -3.62 kcal/mol) compared to positive controls. Mimosine, L-noradrenaline, octopamine, and L-theanine had toxicity classes of IV, II, IV, and IV, respectively. Conclusion: Octopamine shows the best potential as SARS-CoV-2 spike (S2) glycoprotein inhibitor. However, this compound also poses several toxicity risks and therefore, needs a more elaborate considera-tion upon using. There are still no specific treatments for coronavirus disease (COVID-19) until present days. Several studies have been conducted to determine whether herbal medicine can be an option to be used as a definitive medicine for COVID-19. S2 subunit of spike protein which is responsible for SARS-CoV-2 entry to the host cell, is a potential drug target to inhibit the viral infection. In this study, we aimed to find some compounds from the HerbalDB database that have potential as SARS-CoV-2 spike (S2) glycoprotein inhibitor.

Chelerythrine inhibits HSV-1 infection by suppressing virus binding to the cells

ObjectiveHerpes simplex virus type 1 (HSV-1) is a worldwide pathogen and mainly puts immunocompromised patients at higher risk of various complications. This research aims to demonstrate the antiviral activity of chelerythrine (CHE), an alkaloid compound derived from the traditional Chinese medicine Mcleaya cordata, towards the HSV-1 F strain. MethodsBioassay-guided fractionation was carried out in order to isolate the antiviral compound CHE. The plaque reduction assay was used to evaluate the antiviral activity of CHE. The combination of quantitative PCR (qPCR) and western blot was used to elucidate whether CHE interferes with viral DNA synthesis, followed by time-of-addition assay to examine which steps of HSV-1 infection CHE affected. The immunofluorescent assay and transmission electron microscopy assay were performed to further analyze the intracellular localization and the ultrastructural features of HSV-1 virions with the treatment of CHE. The binding affinity in CHE to targeted viral glycoprotein was investigated by surface plasmon resonance (SPR) assay and docking simulation. ResultsCHE displayed a significant antiviral effect against HSV-1 at 0.312 to 2.5 µM with a viral titer reduction of 29.38% to 61.02%. CHE had an EC50 value of 1.78 µM against HSV-1, and the selectivity index (SI) for CHE was 4.07. Additionally, CHE inhibits ICP0 expression in the immediate early stage of infection, which further affected the early stage of infection, and subsequent viral replication. Results of the SPR assay showed that CHE had strong-binding affinity to gB with a KD value of 7.80 × 107 M, while gD with a value of 1.18 × 106 M. TEM revealed that CHE can clearly inhibit the virion release. In silico experiments further confirmed that CHE had low binding free energy in the activity sites of gB and gD. ConclusionThis study revealed that CHE directly targeted viral gB or gD as a potent glycoprotein inhibitor, which suggested that CHE could be a virus entry inhibitor for HSV-1 treatment.

Molecular docking and dynamics studies for the identification of Nipah virus glycoprotein inhibitors from Indian medicinal plants

The infection by Nipah Virus (NiV), a zoonotic paramyxovirus, is fatal and several outbreaks have been reported in humans in various countries. No effective vaccines or drugs are developed till date to control this infection. The NiV-Glycoprotein (NiV-G) is one of the essential proteins for viral entry by binding to the Ephrin-B receptors. The present study screens the potential phytocompounds that can target NiV-G and thereby inhibit the viral entry to human. Computer-aided virtual screening of 1426 phytocompounds from various medicinal plants was carried out to investigate their efficacy as potential therapeutics. Ribavirin, the currently used drug, was also docked to compare the docking score and intermolecular interactions between ligand and target protein. Further, molecular dynamics simulations and MM-PBSA binding free energy calculations were performed to understand the stability of the docked complexes. Radius of gyrations and Solvent Accessible Surface Area were also performed to evaluate the compactness and solvent behaviour of ligand-receptor complexes during the 100 ns simulation. Our analysis revealed that the alkaloid, Serpentinine, has the highest potency to block NiV-G with favourable binding. Communicated by Ramaswamy H. Sarma

BC-11 is a covalent TMPRSS2 fragment inhibitor that impedes SARS-CoV-2 host cell entry.

Host cell entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is facilitated via priming of its spike glycoprotein by the human transmembrane protease serine 2 (TMPRSS2). Although camostat and nafamostat are two highly potent covalent TMPRSS2 inhibitors, they nevertheless did not hold promise in COVID-19clinical trials, presumably due to their short plasma half-lives. Herein, we report an integrative chemogenomics approach based on computational modeling and in vitro enzymatic assays, for repurposing serine-targeted covalent inhibitors. This led to the identification of BC-11 as a covalent TMPRSS2 inhibitor displaying a unique selectivity profile for serine proteases, ascribable to its boronic acid warhead. BC-11 showed modest inhibition of SARS-CoV-2 (omicron variant) spike pseudotyped particles in a cell-based entry assay, and a combination of BC-11 and AHN 1-055 (a spike glycoprotein inhibitor) demonstrated better viral entry inhibition than either compound alone. Given its low molecular weight and good activity against TMPRSS2, BC-11 qualifies as a good starting point for further structural optimizations.

Synthesis and in silico study of chenodeoxycholic acid and its analogues as an alternative inhibitor of spike glycoprotein of SARS-CoV-2

COVID-19, caused by SARS-CoV-2, is a viral infection that has generated one of the most significant health problems in the world. Spike glycoprotein is a crucial enzyme in viral replication and transcription mediation. There are reports in the literature on using bile acid in the fight against this virus through in vitro tests. This work presents the synthesis of nine chenodeoxycholic acid derivatives (1-9), which were prepared by oxidation, acetylation, formylation, and esterification reactions, and the analogs 6-9 have not yet been reported in the literature and the possibility of conducting an in silico study of bile acid derivatives as a therapeutic alternative to combat the virus using glycoprotein as a macromolecular target. As a result, five compounds (1, 6-9) possessed favorable competitive interactions with the lowest energies compared to the native ligand (BLA), and the highlighted compound 9 got the best scores. At the same time, analog 1 presented the best ADME filter result. Molecular dynamics also simulated these compounds to verify their stability within the active protein site to seek new therapeutic propositions to fight against the pandemic. Physical and spectroscopic data have fully characterized all the compounds. Communicated by Ramaswamy H. Sarma