Small molecule therapeutics to destabilize the ACE2-RBD complex: A molecular dynamics study

The ongoing coronavirus disease 19 (COVID-19) pandemic has infected millions of people, claimed hundreds of thousands of lives, and made a worldwide health emergency. Understanding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mechanism of infection is crucial in the development of potential therapeutics and vaccines. The infection process is triggered by direct binding of the SARS-CoV-2 receptor-binding domain (RBD) to the host-cell receptor angiotensin-converting enzyme 2 (ACE2). Many efforts have been made to design or repurpose therapeutics to deactivate the RBD or ACE2 and prevent the initial binding. In addition to direct inhibition strategies, small chemical compounds might be able to interfere and destabilize the metastable, prefusion complex of ACE2-RBD. This approach can be employed to prevent the further progress of virus infection at its early stages. In this study, molecular docking was employed to analyze the binding of two chemical compounds, SSAA09E2 and Nilotinib, with the druggable pocket of the ACE2-RBD complex. The structural changes as a result of the interference with the ACE2-RBD complex were analyzed by molecular dynamics simulations. Results show that both Nilotinib and SSAA09E2 can induce significant conformational changes in the ACE2-RBD complex, intervene with the hydrogen bonds, and influence the flexibility of proteins. Moreover, essential dynamics analysis suggests that the presence of small molecules can trigger large-scale conformational changes that may destabilize the ACE2-RBD complex.     

Effects of alpha lipoic acid and its derivative “andrographolid-lipoic acid-1” on ulcerative colitis: A systematic review with meta-analysis of animal studies

We aimed to review and meta-analyze the inflammatory and oxidative factors following alpha lipoic acid (ALA) and its derivative “andrographolid-lipoic acid-1” (AL-1) in ulcerative colitis (UC). ALA plays an important role in scavenging intracellular radicals and inflammatory elements. AL-1 is found in herbal medicines with potent anti-inflammatory properties. Data were collected from the Google Scholar, PubMed, Scopus, Evidence-based medicine/clinical trials, and Cochrane library database until 2017, which finally resulted in 22 animal studies (70 rats and 162 mice). The beneficial effects of ALA or AL-1 on the most important parameters of UC were reviewed; also, studies were considered separately in mice and rats. Administration of ALA and AL-1 significantly reduced the tumor necrosis factor-α level compared with the controls, while data were not noteworthy in the meta-analysis (mean differences = −18.57 [95% CI = −42.65 to 5.51], P = 0.13). In spite of insignificant decrease in meta-analysis outcomes (differences = 6.92 [95% CI = −39.33 to 53.16], P = 0.77), a significant reduction in myeloperoxidase activity was shown following ALA or AL-1 treatment compared with the controls. Despite significant differences in each study, we had to exclude some studies to homogenize data for meta-analyzing as they showed insignificant results. Interleukin 6, cyclooxygenase-2, glutathione, malondialdehyde, superoxide dismutase, histopathological score, macroscopic and microscopic scores, disease activity index, body weight change, and colon length were also reviewed. Most studies have emphasized on significant positive effects of ALA and AL-1. Comprehensive clinical trials are obligatory to determine the precious position of ALA or AL-1 in the management of UC.     

Site-specific loss of acetylation upon phosphorylation of histone H3

Post-translational modification of histones is a central aspect of gene regulation. Emerging data indicate that modification at one site can influence modification of a second site. As one example, histone H3 phosphorylation at serine 10 (Ser(10)) facilitates acetylation of lysine 14 (Lys(14)) by Gcn5 in vitro (, ). In vivo, phosphorylation of H3 precedes acetylation at certain promoters. Whether H3 phosphorylation globally affects acetylation, or whether it affects all acetylation sites in H3 equally, is not known. We have taken a genetic approach to this question by mutating Ser(10) in H3 to fix either a negative or a neutral charge at this position, followed by analysis of the acetylation states of the mutant histones using site-specific antibodies. Surprisingly, we find that conversion of Ser(10) to glutamate (S10E) or aspartate (S10D) causes almost complete loss of H3 acetylation at lysine 9 (Lys(9)) in vivo. Acetylation of Lys(9) is also significantly reduced in cells bearing mutations in the Glc7 phosphatase that increase H3 phosphorylation levels. Mutation of Ser(10) in H3 and the concomitant loss of Lys(9) acetylation has minimal effects on expression of a Gcn5-dependent reporter gene. However, synergistic growth defects are observed upon loss of GCN5 in cells bearing H3 Ser(10) mutations that are reminiscent of delays in G(2)/M progression caused by combined loss of GCN5 and acetylation site mutations. Together these results demonstrate that H3 phosphorylation directly causes site-specific and opposite changes in acetylation levels of two residues within this histone, Lys(9) and Lys(14), and they highlight the importance of these histone modifications to normal cell functions.     

Investigation into Stability of Poly(Vinyl Alcohol)-Based Opadry® II Films

Poly(vinyl alcohol) (PVA)-based formulations are used for pharmaceutical tablet coating with numerous advantages. Our objective is to study the stability of PVA-based coating films in the presence of acidic additives, alkaline additives, and various common impurities typically found in tablet formulations. Opadry® II 85F was used as the model PVA-based coating formulation. The additives and impurities were incorporated into the polymer suspension prior to film casting. Control and test films were analyzed before and after exposure to 40°C/75% relative humidity. Tests included film disintegration, size-exclusion chromatography, thermal analysis, and microscopy. Under stressed conditions, acidic additives (hydrochloric acid (HCl) and ammonium bisulfate (NH₄HSO₄)) negatively impacted Opadry® II 85F film disintegration while NaOH, formaldehyde, and peroxide did not. Absence of PVA species from the disintegration media corresponded to an increase in crystallinity of PVA for reacted films containing HCl. Films with NH₄HSO₄ exhibited slower rate of reactivity and less elevation in melting temperature with no clear change in melting enthalpy. Acidic additives posed greater risk of compromise in disintegration of PVA-based coatings than alkaline or common impurities. The mechanism of acid-induced reactivity due to the presence of acidic salts (HCl vs. NH₄HSO₄) may be different.     

Evaluation of the Performance Characteristics of Bilayer Tablets: Part I. Impact of Material Properties and Process Parameters on the Strength of Bilayer Tablets

Bilayer tableting technology has gained popularity in recent times, as bilayer tablets offer several advantages over conventional tablets. There is a dearth of knowledge on the impact of material properties and process conditions on the performance of bilayer tablets. This paper takes a statistical approach to develop a model that will determine the effect of the material properties and bilayer compression process parameters on the bonding strength and mode of breakage of bilayer tablets. Experiments were carried out at pilot scale to simulate the commercial manufacturing conditions. As part of this endeavor, a seven-factor half-fraction factorial (2⁷⁻¹) design was executed to study the effect of bilayer tablet compression process factors on the bonding strength of bilayer tablets. Factors studied in this work include: material properties (plastic and brittle), layer ratio, dwell time, layer sequence, first- and second-layer forces, and lubricant concentration. Bilayer tablets manufactured in this study were tested using the axial tester, as it considers both the interfacial and individual layer bonding strengths. Responses of the experiments were analyzed using PROC GLM of SAS (SAS Institute Inc, Cary, North Carolina). A model was fit using all the responses to determine the significant interactions (p < 0.05). The results of this study indicated that nature of materials played a critical role on the strength of bilayer compacts and also on mode of fracture. Bilayer tablets made with brittle materials in both the layers are strongest, and fracture occurred in the first layer indicating that interface is stronger than layers. Significant interactions were observed between the selected factors and these results will provide an insight into the interplay of material properties, process parameters, and lubricant concentration on the bonding strength and mode of breakage of bilayer tablets.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-012-9845-9) contains supplementary material, which is available to authorized users.KEY WORDS: axial tester, bilayer tablets, DOE, interfacial strength, process parameters     

Some characteristics of rabbit muscle phosphofructokinase-1 inhibition by ascorbate

These studies relate to a working hypothesis that glycogen storage is facilitated in resting muscle by inhibiting glycolysis via inhibition of LDH, AK, and PFK-1 by ascorbate; when muscle is active, these isozymes combine with muscle proteins and are released and protected from inhibition by ascorbate and glycolysis proceeds. Focus in these studies is on the ability of G-actin and aldolase to prevent PFK-1 inhibition by ascorbate. We found that inhibition by ascorbate was PFK-1 concentration dependent; ascorbate does not inhibit above 200 nM PFK-1. We conclude that ascorbate inhibits PFK-1 dimers (and perhaps monomers) but not PFK-1 tetramers. Separation of PFK-1 dimers from tetramers was achieved with centrifugal filter devices and differences in their sensitivity to ascorbate inhibition were demonstrated. Some comparisons are made with attributes of AK inhibitions by ascorbate that, like PFK-1, are also enzyme concentration dependent. Discussions relate findings to cellular infrastructure and the role of ascorbate in glycogen synthesis.     

Suicidal Membrane Repair Regulates Phosphatidylserine Externalization during Apoptosis

One of the hallmarks of apoptosis is the redistribution of phosphatidylserine (PS) from the inner-to-outer plasma membrane (PM) leaflet, where it functions as a ligand for phagocyte recognition and the suppression of inflammatory responses. The mechanism by which apoptotic cells externalize PS has been assumed to involve “scramblases” that randomize phospholipids across the PM bilayer. These putative activities, however, have not been unequivocally proven to be responsible for the redistribution of lipids. Because elevated cytosolic Ca²⁺ is critical to this process and is also required for activation of lysosome-PM fusion during membrane repair, we hypothesized that apoptosis could activate a “pseudo”-membrane repair response that results in the fusion of lysosomes with the PM. Using a membrane-specific probe that labels endosomes and lysosomes and fluorescein-labeled annexin 5 that labels PS, we show that the appearance of PS at the cell surface during apoptosis is dependent on the fusion of lysosomes with the PM, a process that is inhibited with the lysosomotrophe, chloroquine. We demonstrate that apoptotic cells evoke a persistent pseudo-membrane repair response that likely redistributes lysosomal-derived PS to the PM outer leaflet that leads to membrane expansion and the formation of apoptotic blebs. Our data suggest that inhibition of lysosome-PM fusion-dependent redistribution of PS that occurs as a result of chemotherapy- and radiotherapy-induced apoptosis will prevent PS-dependent anti-inflammatory responses that preclude the development of tumor- and patient-specific immune responses.There is increasing evidence that damaged plasma membranes (PM)² trigger an emergency Ca²⁺-dependent exocytotic repair response that patches the affected area by adding lysosome-derived membranes at the cell surface disruption site (1–5). Because high cytosolic Ca²⁺ concentrations trigger lysosome-PM fusion, the elevated cytosolic Ca²⁺ levels characteristic to apoptotic cells may also evoke a pseudo-repair mechanism that promotes lysosome-PM fusion. Indeed, similar to normal emergency repair responses, apoptosis is characterized by the appearance of organelle proteins and lipids at the PM surface (6–8). One critical distinction between the apoptotic and physiologic repair processes is the preservation of membrane lipid asymmetry. In normal cells, any perturbation in PS sidedness is corrected by restoration of basal cytosolic [Ca²⁺], reactivation of the Ca²⁺-inhibited aminophospholipid translocase (9, 10), and subsequent facilitated transport of PS back to the inner membrane leaflet of the cell. In apoptotic cells, however, persistent high cytosolic [Ca²⁺] precludes reactivation of the aminophospholipid translocase, and the redistributed PS remains in the outer membrane leaflet (11). The apparent similarities in these processes combined with observations that apoptotic cells express PS at the cell surface prompted us to investigate whether lysosome to PM fusion plays a role in the redistribution of PS during apoptosis.     

Reduction of diabetes-induced oxidative stress by phosphodiesterase inhibitors in rats

Increased oxidative stress has been suggested to be involved in the pathogenesis and progression of diabetic tissue damage. The aim of this study was to investigate the effect of different phosphodiesterase inhibitors on lipid peroxidation and total antioxidant capacity (TAC) of plasma in streptozotocin-induced diabetic rats (Rattus norvegicus). Rats became diabetic by a single administration of streptozotocin (STZ, 45 mg/kg). The effects of 15-days treatment by milrinone, sildenafil, and theophylline as cyclic-AMP and -GMP phosphodiesterase inhibitors (PDEIs) on diabetes-induced oxidative stress were studied. The levels of glucose, malonedialdehyde (MDA) the by product of lipid peroxides, and TAC (FRAP test) were estimated in plasma of control and experimental groups of rats. A significant increase in the levels of plasma glucose, and MDA and a concomitant decrease in the levels of TAC were observed in diabetic rats. These alterations were reverted back to near normal level after the treatment with PDEIs. Treatment of diabetic rats by PDEIs reduced MDA levels and increased TAC in the order of milrinone>sildenafil>theophylline. In conclusion, the present investigation show that PDIS possesses antioxidant activities, which may be attributed to their enhancing effect on cellular cyclic nucleotides contributing to the protection against oxidative stress in streptozotocin-induced diabetes. Exact mechanism of protective actions of cAMP- and cGMP-phosphodiesterase remains to be elucidated by further studies. This finding may suggest a place for PDEIs in maintaining health in diabetes.     

Regulated externalization of phosphatidylserine at the cell surface: implications for apoptosis

The regulated loss of plasma membrane phosphatidylserine (PS) asymmetry is critical to many biological processes. In particular, the appearance of PS at the cell surface, a hallmark of apoptosis, prepares the dying cell for engulfment and elimination by phagocytes. While it is well established that PS externalization is regulated by activation of a calcium-dependent phospholipid scramblase activity in concert with inactivation of the aminophospholipid translocase, there is no evidence indicating that these processes are triggered and regulated by apoptotic regulatory mechanisms. Using a novel model system, we show that PS externalization is inducible, reversible, and independent of cytochrome c release, caspase activation, and DNA fragmentation. Additional evidence is presented indicating that the outward movement of plasma membrane PS requires sustained elevation in cytosolic Ca2+ in concert with inactivation of the aminophospholipid translocase and is inhibited by calcium channel blockers.