Computational Method on Hydroxychloroquine and Azithromycin for SARS-CoV-2: Binding Affinity Studies

World is facing a new pandemic called covid-19SARS-CoV-2) since a year ago. Unfortunately there is no treatment for Covid 19 nowadays as well as no potential therapies has been developed to overcome from coronavirus pandemic. Some potential drug molecules with combination have ability to respond for covid19 virus. From the research it was found that the reduction of viral load can be treated with hydroxychloroquine and azithromycin combination. We evaluate the mode of interactions of hydroxychloroquine and azithromycin with the dynamic site of SARS-CoV-2 coronavirus main protease. Molecular Structure-based computational approach viz. molecular docking simulations were performed to scale up their affinity and binding fitness of the docked complex of novel SARS-CoV-2 coronavirus protease and hydroxychloroquine and azithromycin. The natural inhibitor N3 of novel SARS-CoV-2 coronavirus protease were exhibited highest affinity in terms of MolDock score (-167.203Kcal/mol), and hydroxychloroquine was found with lowest target affinity (-55.917 Kcal/mol).The amino acid residue cysteine 145 and histidine 41 is bound covalently and formed hydrogen bond interaction with SARS-CoV-2 inhibitor known as inhibitor N3 as such, hydroxychloroquine and azithromycin also formed hydrogen bond interaction. The binding patterns of the inhibitor N3 of SARS-CoV-2 coronavirus main protease could be used as a guideline for medicinal chemist to explore their SARS-CoV-2 inhibitory potential. Copyright © RJPT All right reserved.

A Spotlight on the Development, Pharmaceutical Trends, Innovations and Patents of Nirmatrelvir (Paxlovid™)

Paxlovid™ is a combination of Nirmatrelvir and Ritonavir antiviral pills with good oral bioavailability. In clinical studies, treatment of the patients infected with SARS-CoV-2 with Paxlovid™ within three to five days of the appearance of symptoms significantly reduced the hospitalization rate as well as mortality. It is the first oral antiviral treatment for the COVID-19 which received USFDA approval for EUA on 22nd December, 2021. Nirmatrelvir inhibits the replication of SARS-CoV-2 while another antiviral drug, Ritonavir, is given in combination to enhance the bioavailability of Nirmatrelvir. Molecular interaction studies have shown that Nirmatrelvir binds covalently with the catalytic triad of the active site of the viral protease enzyme (3CLPRO). It, therefore, acts by stopping the SARS-CoV-2 replication by its ability to block the translation of the viral genetic materials. Research studies conducted have proven the efficacy of this oral anti-viral drug in mild to moderate COVID-19 patients beside its ease of oral administration and good oral bioavailability. Alternative synthetic methods to scale up the synthesis of this potent molecule are needed to reduce the treatment cost of the COVID-19. Extensive clinical research on a larger group population is also underway for ensuring the safety and efficacy of this medication in the battle against the COVID-19 pandemic.

HEMORRHAGIC PLEURAL EFFUSION IN A PATIENT WITH CHRONIC LYMPHOCYTIC LEUKEMIA ON IBRUTINIB THERAPY

SESSION TITLE: Problems in the Pleura Case Posters 1 SESSION TYPE: Case Report Posters PRESENTED ON: 10/17/2022 12:15 pm - 01:15 pm INTRODUCTION: Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (Btk), approved for treatment of a variety of B-cell malignancies, including chronic lymphocytic leukemia (CLL). There is an association of increased risk of bleeding with ibrutinib due to platelet dysfunction caused by the medication. Bleeding is usually non-life threating such as subcutaneous or mucosal bleeding, epistaxis, and ecchymosis. But major bleeding has been reported such as intracranial hemorrhage and gastrointestinal hemorrhage. Thoracic complications from ibrutinib are rare. Below is a case report discussing a hemorrhagic pleural effusion thought to be caused by Ibrutinib. CASE PRESENTATION: Patient is a 78-year-old male initially diagnosed with CLL on flow cytometry showing a low-grade B cell lymphoproliferative process. Patient was monitored by Hematology and when kappa light chain numbers began to rise, a bone marrow biopsy was performed showing 90% infiltration of the marrow with lymphoid cells. Patient was started on Ibrutinib therapy and responded well to treatment. A year after starting therapy, patient presented to the emergency room with increased shortness of breath and fatigue. Patient was found to be COVID-19 positive and chest x-ray showed a large right sided pleural effusion. Thoracentesis was performed draining 1650cc of bloody fluid. Fluid studies revealed a lymphocytic effusion with RBC count 1,185375, WBC of 1751. Cultures and cytology were negative. On further history, patient was without recent trauma or surgery, CTA chest was negative for pulmonary embolism. QuantiFERON Gold test was negative, indicating low likelihood of tuberculosis. Patient was not on any antiplatelet or systemic anticoagulation medications. Ibrutinib therapy was held during hospitalization and pleural effusion did not reaccumulate. Patient passed away during hospital stay secondary to respiratory failure due to COVID-19. DISCUSSION: Ibrutinib is an orally bioavailable bruton tyrosine kinase inhibitor (BTKi) and forms an irreversible covalent bound to BTK at the Cysteine-481 residue. Ibrutinib predisposes to bleeding by inhibiting BTK and Tec, which play a role in the inhibitory signaling pathway of platelet collagen receptors such as glycoprotein VI (GP VI) and C-type lectin-like receptor 2 (CLEC-2). Our patient had no other risk factors for developing a hemorrhagic effusion. CLL itself can cause malignant effusions, one study found the incidence of malignant effusions among patients with CLL to be 9%, but the effusion was noted to be serous or serosanguinous and there was pleural involvement in all patients which was not the case in our patient. CONCLUSIONS: There is currently a minimal amount of data to guide clinicians regarding the use of ibrutinib in patients at high risk of bleeding or on anticoagulant or antiplatelet therapy. It is important to realize bleeding complications related to ibrutinib therapy can occur. Reference #1: Shatzel JJ, Olson SR, Tao DL, McCarty OJT, Danilov AV, DeLoughery TG. Ibrutinib-associated bleeding: pathogenesis, management and risk reduction strategies. J Thromb Haemost. 2017;15(5):835-847. doi:10.1111/jth.13651 Reference #2: Burger JA, Tedeschi A, Barr PM, et al. Ibrutinib as Initial Therapy for Patients with Chronic Lymphocytic Leukemia. N Engl J Med. 2015;373(25):2425-2437. doi:10.1056/NEJMoa1509388 Reference #3: Paydas S. Management of adverse effects/toxicity of ibrutinib. Crit Rev Oncol Hematol. 2019;136:56-63. doi:10.1016/j.critrevonc.2019.02.001 DISCLOSURES: No relevant relationships by fatima ali No relevant relationships by Joan Wiley

PURIFICATION OF ANTI-RBD ANTIBODIES OBTAINED FROM LLAMA (Lama glama) FOR POTENTIAL COVID-19 THERAPY

Despite worldwide efforts to develop different treatments for SARS-CoV-2 COVID-19, the situation remains critical, requiring rapid and effective strategies. In this regard, antibodies (Ab) have demonstrated clinical potential. Among them, camelid nanoAb (VHH) arise as a possible alternative, as they recognize epitopes which are inaccessible to conventional Ab. Further advantages of VHH are their small size, high solubility, high stability, and resistance to low pH. The aim of this work is to describe a purification scheme of different isotypes of anti-SARS-CoV-2 immunoglobulin G (IgG) produced after immunizing two llamas (Lama glama). To achieve this, plasma was injected into an affinity chromatographic column (Protein G), and the resulting fractions were analyzed by SDS-PAGE under non-reducing conditions. The anti-RBD titers were determined by an “in house” ELISA, reaching titers of 52000 and 13000 for IgG1 and IgG3 fractions, respectively. Subsequently, an affinity column (HiTrap NHS-activated) was prepared to separate monospecific anti-RBD polyclonal Ab. RBD produced in our laboratory was covalently coupled to this column, achieving a coupling efficiency of 97%. Different isotypes of monospecific anti-RBD Ab (IgG1: 140 kDa and IgG3: 95 kDa) were obtained. IgG3 represent the starting point for obtaining VHH and/or evaluating their potential use as a therapeutic or preventive alternative, which represents a notable regional contribution in the fight against COVID-19.

Targeting brain-resident viruses across the blood-brain barrier in vitro using peptideporphyrin conjugates

The recent SARS-CoV-2 pandemic brought awareness to the permanent dangers of viral infections and outbreaks. Beyond its inherent infections, several viruses such as Dengue (DENV), Zika (ZIKV), HIV and even SARS-CoV-2 have the potential to infect the brain, causing more aggressive and irreversible injuries. These brain infections are particularly hard to treat not only because the number of efficient antiviral drugs against these viruses is scarce, but also due to the restrictive permeability of the blood- brain barrier (BBB) that hinders brain drug-intake. To overcome these issues, we designed peptide-drug conjugates formed by covalent attachment of a BBB peptide shuttle and a broad-spectrum antiviral porphyrin drug. We synthesized eighteen novel peptide-porphyrins conjugates (PPCs) and tested their activity in vitro, both in BBB-translocation and antiviral capacity against DENV, ZIKV, HIV and SARS-CoV-2. Cytotoxicity towards pharmacologic relevant cell lines was also studied. After careful fine-tuning of the on-resin synthetic chemistry, DIC/Oxyma coupling has emerged as preferred method, bearing a 99% conjugation yield. Ten PPCs inactivate at least two different viruses in vitro, a selection criterion for further evaluation, with IC50s ranging between 0.5 to 33 lM. Although all ten PPCs efficiently translocate the cellular BBB model in vitro, a set of seven stand out as the most druggable since they are not cytotoxic towards all cell lines tested. Overall, peptide-porphyrin conjugation shows to be an innovative and promising strategy to treat viral brain infections.

Repurposing FDA-approved Drugs Targeting SARS-CoV2 3CLpro: A Study by Applying Virtual Screening, Molecular Dynamics, MM-PBSA Calculations and Covalent Docking

Background: Since the end of 2019, the etiologic agent SAR-CoV-2 responsible for one of the most significant epidemics in history has caused severe global economic, social, and health damages. The drug repurposing approach and application of Structure-based Drug Discovery (SBDD) using in silico techniques are increasingly frequent, leading to the identification of several molecules that may represent promising potential. Methods: In this context, here we use in silico methods of virtual screening (VS), pharmacophore modeling (PM), and fragment-based drug design (FBDD), in addition to molecular dynamics (MD), molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) calculations, and covalent docking (CD) for the identification of potential treatments against SARS-CoV-2. We initially validated the docking protocol followed by VS in 1,613 FDA-approved drugs obtained from the ZINC database. Thus, we identified 15 top hits, of which three of them were selected for further simulations. In parallel, for the compounds with a fit score value ≤ of 30, we performed the FBDD protocol, where we designed 12 compounds. Results: By applying a PM protocol in the ZINC database, we identified three promising drug candidates. Then, the 9 top hits were evaluated in simulations of MD, MM-PBSA, and CD. Subsequently, MD showed that all identified hits showed stability at the active site without significant changes in the pro-tein's structural integrity, as evidenced by the RMSD, RMSF, Rg, SASA graphics. They also showed interactions with the catalytic dyad (His41 and Cys145) and other essential residues for activity (Glu166 and Gln189) and high affinity for MM-PBSA, with possible covalent inhibition mechanism. Conclusion: Finally, our protocol helped identify potential compounds wherein ZINC896717 (Zafir-lukast), ZINC1546066 (Erlotinib), and ZINC1554274 (Rilpivirine) were more promising and could be explored in vitro, in vivo, and clinical trials to prove their potential as antiviral agents.

Safety and Efficacy of Evobrutinib, a Bruton's Tyrosine Kinase Inhibitorin Relapsing Multiple Sclerosis Over 2.5 Years of The Open-Label Extension to a Phase II Trial

Objective: Report the safety and efficacy of evobrutinib over 2.5 years in an open-label extension (OLE). Background: Evobrutinib, a covalent, blood-brain barrier-penetrating Bruton's tyrosine kinase inhibitor, was well tolerated and effective at reducing gadolinium-enhancing lesions in a double-blind, randomized phase II trial in patients with relapsing multiple sclerosis (pwRMS;NCT02975349). Design/Methods: In the 48-week (W) double-blind period (DBP), pwRMS (n=267) received placebo (switched to evobrutinib 25mg once-daily at W24), evobrutinib 25mg once-daily, 75mg once-daily, or 75mg twice-daily, or open-label dimethyl fumarate (DMF;240mg twice-daily). At W48 patients could enter the OLE (DMF: 4-8W washout);evobrutinib 75mg once-daily (median ~48W) then 75mg twice-daily. The latest available OLE data are now reported. Results: Of 267 DBP patients, 213 (80%) entered the OLE;164 (61%) completed ≥132W of OLE treatment. Treatment-emergent adverse events (TEAEs) were reported by 165/213 patients (77.5%);59 (27.7%) had a treatment-related TEAE. Six serious TEAEs were deemed treatment-related. Severe/opportunistic infections (≥Grade 3) were reported by 9/213 patients (4.2%);3 were fatal (Covid-19 pneumonia [n=2] and E. coli sepsis [n=1];not considered treatment-related). At OLE W120, most patients had IgG (91%), IgA (88%) and IgM (82%) within normal ranges. Overall mean CD19+ B cells levels were 0.218×10 cells/mL (OLE baseline) and 0.122×10 cells/mL (OLE W96). ALT/AST elevations were observed only in patients previously receiving DMF/evobrutinib 25mg and occurred within 12W of OLE initiation. Amylase/lipase increases occurred in 6 (2.8%)/24 (11.3%) patients, but without clinical signs and symptoms. Based on all available OLE data, ARR was 0.12 (95%CI 0.07-0.20) for patients receiving 75mg twice-daily in the DBP. 6 6 Conclusions: Evobrutinib safety and efficacy data over 2.5 years in pwRMS continue to show acceptable tolerability, with no new safety signals, and maintained efficacy.

Pharmacologic inhibition of SUMOactivating enzyme potentiates interferon response and T cell-mediated anti-tumor immunity in chronic lymphocytic leukemia (CLL) and lymphoma models

Introduction: CLL is characterized by deficient immunity which clinically manifests as an increased predisposition toward malignancies and infectious complications. T-cells from patients with CLL exhibit a skewed repertoire with a predominance of Tregs as well as impaired immune synapse formation and cytotoxic function. Unlike chemotherapy, novel targeted agents may have beneficial immunomodulatory effects, which may be particularly relevant in the COVID-19 era. Small ubiquitin-like modifier (SUMO) family proteins regulate a variety of cellular processes, including nuclear trafficking, gene transcription, and cell cycle progression, via post-translational modification of target proteins. Sumoylation regulates NFjB signaling, IFN response, and NFAT activation, processes indispensable in immune cell activation. Despite this, the role of sumoylation in T cell biology in the context of cancer is not known. TAK-981 is a small molecule inhibitor of the SUMO-activating enzyme (SAE) that forms a covalent adduct with an activated SUMO protein, thereby preventing its transfer to the SUMO-conjugating enzyme (Ubc9). Here, we investigated the immunomodulatory effects of TAK-981 in CLL. Methods: T cells from patients with CLL were purified using Dynabeads. Activation, proliferation, and apoptosis of CD3+ T cells were studied following T-cell receptor engagement (TCR;aCD3/CD28) with/without 0-1 lM TAK-981. Cytokines were measured after in vitro stimulation. For polarization assays, FACS-sorted naïve CD4+ T cells were cultured for 7 days in control or differentiation media. For gene expression profiling (GEP;Clariom S), RNA was harvested after 3 and 24 h of TCR engagement from FACS-sorted naïve CD4+ T cells. For in vivo immunization experiments, CD4+KJ1-26+ cells were inoculated IV into BALB/cJ mice. Mice received 100 mg IV ovalbumin ± R848 followed by TAK-981 7.5 mg/kg or vehicle control IV twice weekly for 10 days before spleen collection. Both recipient and transplanted splenocytes were analyzed. For analysis of tumor-infiltrating lymphocytes (TILs), BALB/c mice were injected with 1×106 A20 lymphoma cells and treated as above. TAK-981 was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA). Results: T cells from patients with CLL demonstrated high baseline protein sumoylation that slightly increased following TCR engagement. Treatment with TAK-981 significantly downregulated SUMO1 and SUMO2/3-modified protein levels, yet did not disrupt early TCR signaling as evidenced by sustained ZAP70, p65/NFjB, and NFAT activation detected by immunoblotting, immunocytochemistry, and GEP. Treatment with TAK-981 resulted in dose-dependent upregulation of the early activation marker CD69 in CD4+ T cells following 72 and 96 h of TCR stimulation vs. control. Meanwhile, the expression of CD25, HLA-DR, and CD40L was delayed in the presence of TAK-981. Interestingly, CD38, an IFN response target, was induced 2-fold in TAK-981-treated cells after 24 h and persisted at high levels at subsequent timepoints. T cell proliferation was reduced in the presence of high (1 lM) but not low/intermediate concentrations of TAK-981, accompanied by reduced S phase entry and decreased synthesis of IL- 2. However, T cells did not undergo apoptosis under those conditions. Targeting SAE in either control or Th1/Treg polarizing conditions facilitated an increase in IFNc and loss of FoxP3 expression (accompanied by decreased IL-2/STAT5), suggesting a shift toward Th1 and away from Treg phenotype, respectively. GEP (Reactome, GSEA) confirmed a dramatically upregulated IFN response in TAK-981-treated CD4+ naïve T cells. Furthermore, targeting SAE enhanced degranulation (CD107a), IFNc, and perforin secretion in cytotoxic CD8+ T cells and potentiated T cell cytotoxicity in allogeneic assays with lymphoma cells (OCI-LY3, U2932) as targets. Consistent with our in vitro data, OVA-stimulated transplanted transgenic KJ1-26+ splenocytes, as well as total CD4+ T cells from recipient mice treated with TAK-981 in vivo exhibited a significant reduction in express on of FoxP3 and an increased production of IFNc. In the A20 syngeneic model, treatment with TAK-981 similarly downregulated FoxP3 expression in CD4+ TILs and induced IFNc secretion in CD8+ TILs. Conclusion: Using a combination of in vitro and in vivo experiments, we demonstrate that pharmacologic targeting of sumoylation with TAK-981 does not impair proximal TCR signaling in T cells obtained from patients with CLL, but leads to rebalancing toward healthy immune T cell subsets via induction of IFN response and downmodulation of Tregs. These data provide a strong rationale for continued investigation of TAK-981 in CLL and lymphoid malignancies.

Safety and Efficacy of Acalabrutinib Plus Venetoclax and Rituximab in Patients with Treatment-Naïve (TN) Mantle Cell Lymphoma (MCL)

Background: First-line conventional chemoimmunotherapy in MCL can be improved. Promising results have been seen with Bruton tyrosine kinase inhibitors (BTKis) in combination with venetoclax (V) and an anti-CD20 monoclonal antibody in patients (pts) with relapsed/refractory or TN MCL. Acalabrutinib (A) is a next-generation, highly selective, covalent BTKi currently approved for relapsed/refractory MCL. We report initial safety and efficacy results of the ongoing, multicenter, open-label phase 1b study of A, V, and rituximab (R) (AVR) in TN MCL. Methods: TN MCL pts aged ≥18 y with ECOG PS ≤2 were eligible. Starting on cycle 1 day 1, A was administered at 100 mg BID until disease progression or discontinuation for other reasons. R was administered at 375 mg/m 2 on day 1 of each 28-day cycle for 6 cycles, followed by maintenance every other cycle for pts achieving complete response (CR) or partial response (PR), through cycle 24. Starting on cycle 2 day 1, V was administered via an initial 5-wk ramp-up schedule (20, 50, 100, 200, and 400 mg/d) to 400 mg/d, through cycle 25. Dose-limiting toxicity (DLT) was assessed from cycle 2 day 1 to cycle 3 day 28. Primary endpoint was AVR safety. Secondary endpoints were overall response rate (ORR), duration of response (DOR), and progression-free survival (PFS) per Lugano criteria. Positron-emission tomography (PET)/computed tomography (CT) scans were performed after 3 and 6 cycles and to confirm CR at any time. CT scans were performed after 3, 6, 9, and 12 cycles, and then every 6 cycles. Longitudinal minimal residual disease (MRD) was assessed using the clonoSEQ assay in peripheral blood at PR, CR, every 6 cycles post-CR, and treatment end. Results: 21 pts were enrolled (median age 66 y [range 51-85];ECOG PS ≤1 20 [95%];Ann Arbor stage IV disease 19 [90%];bulky disease >5 cm 7 [33%];intermediate- and high-risk simplified MCL International Prognostic Index scores 11 [52%] and 4 [19%], respectively;blastoid variant 1 [5%];and Ki-67 proliferation index ≥50% 3 [14%]). Fifteen (71%) pts had bone marrow (BM) involvement at baseline. As of March 19, 2021, median time on study was 16 mo (range 8-26.2). Median (range) number of cycles administered was 15 (7-27) for A, 13.5 (5-23) for 400 mg daily V, and 12 (6-15) for R. Seventeen (81%) pts remain on study treatment and 4 (19%) have discontinued (progressive disease: n=1;COVID-19 infection: n=3). No DLTs were observed;V 400 mg daily after ramp-up was the dose chosen for triple therapy. Most common any-grade AEs in ≥20% of pts were diarrhea (13 [62%]), headache (11 [52%]), fatigue (10 [48%]), neutropenia (6 [29%]), paresthesia (6 [29%]), cough (6 [29%]), dyspnea (6 [29%]), myalgia (5 [24%]), dizziness (5 [24%]), and hypoesthesia (5 [24%]). Grade 3/4 AEs in ≥2 pts were neutropenia (5 [24%]) and pneumonia (2 [10%]). Serious any-grade AEs in ≥2 pts were COVID-19 infection (4 [19%]) and pneumonia (2 [10%]). In the 4 pts with COVID-19 infection, the events led to triple-drug discontinuation and death in 3 pts and to dose holds of A and V and event resolution in 1 pt (all considered unrelated to study treatment). Diarrhea led to V dose reduction in 1 pt. AEs led to dose holds in 12 (57%) pts and were associated with A, V, and R in 52%, 48%, and 14%, respectively. Events of clinical interest are shown in Table 1. At the end of cycle 6, ORR was 100%, with CR/PR in 90%/10% by PET/CT alone (11 of the 13 CRs by PET/CT lacked BM confirmation);the CR/PR rate by Lugano criteria with BM confirmation was 38%/62% (Table 2). Median DOR was 19 mo (95% CI 17-not estimable [NE]) overall, and not reached when the 3 pts with COVID-19 deaths were censored. Median PFS and OS were not reached. The 1-y PFS and OS rates were 89% (95% CI 62-97) and 95% (95% CI 71-99), respectively. Treating the 3 COVID-19 deaths as censored, the 1-y PFS rate was 93.8% (95% CI 63.2-99.1). Median time to initial response and best response was 2.8 mo. Twelve of 16 (75%) pts with available MRD results at cycle 6 achieved MRD negativity (10 -6), including 6 pts with

The Se-S Bond Formation in the Covalent Inhibition Mechanism of SARS-CoV-2 Main Protease by Ebselen-like Inhibitors: A Computational Study.

The inhibition mechanism of the main protease (Mpro) of SARS-CoV-2 by ebselen (EBS) and its analog with a hydroxyl group at position 2 of the benzisoselenazol-3(2H)-one ring (EBS-OH) was studied by using a density functional level of theory. Preliminary molecular dynamics simulations on the apo form of Mpro were performed taking into account both the hydrogen donor and acceptor natures of the Nδ and Nε of His41, a member of the catalytic dyad. The potential energy surfaces for the formation of the Se-S covalent bond mediated by EBS and EBS-OH on Mpro are discussed in detail. The EBS-OH shows a distinctive behavior with respect to EBS in the formation of the noncovalent complex. Due to the presence of canonical H-bonds and noncanonical ones involving less electronegative atoms, such as sulfur and selenium, the influence on the energy barriers and reaction energy of the Minnesota hybrid meta-GGA functionals M06, M06-2X and M08HX, and the more recent range-separated hybrid functional wB97X were also considered. The knowledge of the inhibition mechanism of Mpro by the small protease inhibitors EBS or EBS-OH can enlarge the possibilities for designing more potent and selective inhibitor-based drugs to be used in combination with other antiviral therapies.