The structural, functional, and dynamic effect of Tau tubulin kinase1 upon a mutation: A neuro-degenerative hotspot.

Alzheimer's disease (AD) is a progressive disorder that causes brain cells to degenerate and die. AD is one of the common causes of dementia that leads to a decline in thinking, behavioral and social skills that disrupts a person's ability to function independently. Tau-tubulin kinase1 (TTBK1) is a crucial disease regulating AD protein, which is majorly responsible for the phosphorylation and accumulation of tau protein at specific Serine/Threonine residues found in paired helical filaments, suggesting its role in tauopathy. TTBK1 involvement in many diseases and the restricted expression of TTBK1 to the central nervous system (CNS) makes TTBK1 an attractive therapeutic target for tauopathies. The genetic variations in TTBK1 are primarily involved in the TTBK1 pathogenesis. This study highlighted the destabilizing, damaging and deleterious effect of the mutation R142Q on TTBK1 structure through computational predictions and molecular dynamics simulations. The protein deviation, fluctuations, conformational dynamics, solvent accessibility, hydrogen bonding, and the residue-residue mapping confirmed the mutant effect to cause structural aberrations, suggesting overall destabilization due to the protein mutation. The presence of well-defined free energy minima was observed in TTBK1-wild type, as opposed to that in the R142Q mutant, reflecting structural deterioration. The overall findings from the study reveal that the presence of R142Q mutation on TTBK1 is responsible for the structural instability, leading to disruption of its biological functions. The mutation could be used as future diagnostic markers in treating AD.

Detection of truncated isoforms of human neuroserpin lacking the reactive center loop: Implications in noninhibitory role.

Neuroserpin is a serine protease inhibitor expressed mainly in the brain and at low levels in other tissues like the kidney, testis, heart, and spinal cord. It is involved in the inhibition of tissue plasminogen activator (tPA), plasmin, and to a lesser extent, urokinase-type plasminogen (uPA). Neuroserpin has also been shown to plays noninhibitory roles in the regulation of N-cadherin-mediated cell adhesion. It is involved in neuroprotection from seizure and stroke through tPA-mediated inhibition and also through its other protease targets. Mutations in critical domains of neuroserpin lead to its polymerization and neuronal death. In this study, a novel truncated isoform of human neuroserpin was identified in the brain and liver, which was confirmed by reverse transcriptase-PCR and DNA sequencing using exon-specific primers. Structural characterization of novel isoform using MD simulations studies indicated that it lacks the reactive center loop (RCL) but largely maintains its secondary structure fold. The novel truncated variant was cloned, expressed, and purified. A comparative intrinsic fluorescence and 4,4'-bis-1-anilino naphthalene 8-sulfonate studies revealed a decrease in fluorescence emission intensity and a more exposed hydrophobic surface as compared to the reported isoform. However, the novel isoform has lost its ability for tPA inhibition and complex formation. The absence of RCL indicates a noninhibitory role for the truncated isoform, prompting a detailed search and identification of two smaller isoforms in the human brain. With indications of the noninhibitory role of neuroserpin, identifying novel isoforms that appear to be without the tPA recognition domain is significant.

Identification and characterization of a novel isoform of heparin cofactor II in human liver.

Heparin cofactor II (HCII) is predominantly expressed in the liver and inhibits thrombin in blood plasma to influence the blood coagulation cascade. Its deficiency is associated with arterial thrombosis. Its cleavage by neutrophil elastase produces fragment that helps in neutrophil chemotaxis in the acute inflammatory response in human. In the present study, we have identified a novel alternatively spliced transcript of the HCII gene in human liver. This novel transcript includes an additional novel region in continuation with exon 3 called exon 3b. Exon 3b acts like an alternate last exon, and hence its inclusion in the transcript due to alternative splicing removes exon 4 and encodes for a different C-terminal region to give a novel protein, HCII-N. MD simulations of HCII-N and three-dimensional structure showed a unique 51 amino acid sequence at the C-terminal having unique RCL-like structure. The HCII-N protein purified from bacterial culture showed a protein migrating at lower molecular weight (MW 55 kDa) as compared to native HCII (MW 66 kDa). A fluorescence-based analysis revealed a more compact structure of HCII-N that was in a more hydrophilic environment. The HCII-N protein, however, showed no inhibitory activity against thrombin. Due to large conformational variation observed in comparison with native HCII, HCII-N may have alternate protease specificity or a non-inhibitory role. Western blot of HCII purified from large plasma volume showed the presence of a low MW 59 kDa band with no thrombin activity. This study provides the first evidence of alternatively spliced novel isoform of the HCII gene.

Hesperidin-CAMKIV interaction and its impact on cell proliferation and apoptosis in the human hepatic carcinoma and neuroblastoma cells.

Calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a key regulatory molecule of cell signaling, and thereby controls its growth and proliferation, including expression of certain genes. The overexpression of CAMKIV is directly associated with the development of different types of cancers. Hesperidin is abundantly found in citrus fruits and exhibits wide range of pharmacological activities including anti-inflammatory, antibacterial and anticancerous effects. We have investigated binding mechanism of hesperidin with the CAMKIV using molecular docking methods followed by fluorescence quenching and isothermal titration calorimetric assays. An appreciable binding affinity of hesperidin was observed with CAMKIV during fluorescence quenching and isothermal titration calorimetric studies. Efficacy of hesperidin to inhibit the growth of human hepatic carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cancer cell lines were investigated. Hesperidin has significantly reduced the proliferation of HepG2 and SH-SY5Y cells and induces apoptosis by activating the caspase-3-dependent intrinsic pathway through the upregulation of proapoptotic Bax protein. Hesperidin treatment reduces the mitochondrial membrane potential of HepG2 and SH-SY5Y cells. All these observations clearly anticipated hesperidin a potent inhibitor of CAMKIV which may be further exploited a newer therapeutic approach for the management of different cancer types.

Identification of a novel alternatively spliced isoform of antithrombin containing an additional RCL-like loop.

Antithrombin (AT3) is one of the most important inhibitors of blood coagulation proteases that belong to the serpin family of protease inhibitors. In this study, a novel alternatively spliced isoform of AT3 was identified, both at transcript and protein level. This novel transcript contains an additional region in the continuation of exon 3b that was included in the transcript due to use of an alternate 5' splice site. The existence of the novel transcript was confirmed in human brain and liver through RT-PCR. An analysis of the complete transcript indicated that the native reactive centre loop (RCL) of AT3 is maintained; however the novel amino acid sequence projects out as an additional loop as evident from MD simulation studies. A unique amino acid sequence present in the novel isoform was used for the development of polyclonal antibody. The expression of novel isoform was confirmed in human brain and liver tissue using Western blot analysis. Interestingly an alignment of RCL like domain with other inhibitory serpins showed significant similarity with the neuroserpin RCL. To the best of our knowledge, this is the first evidence of alternatively spliced AT3 sequence containing an additional loop and could have physiological relevance.

Evidence of vanillin binding to CAMKIV explains the anti-cancer mechanism in human hepatic carcinoma and neuroblastoma cells.

Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr kinase family, and is associated with different types of cancer and neurodegenerative diseases. Vanillin is a natural compound, a primary component of the extract of the vanilla bean which possesses varieties of pharmacological features including anti-oxidant, anti-inflammatory, anti-bacterial and anti-tumor. Here, we have investigated the binding mechanism and affinity of vanillin to the CAMKIV which is being considered as a potential drug target for cancer and neurodegenerative diseases. We found that vanillin binds strongly to the active site cavity of CAMKIV and stabilized by a large number of non-covalent interactions. We explored the utility of vanillin as anti-cancer agent and found that it inhibits the proliferation of human hepatocyte carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells in a dose-dependent manner. Furthermore, vanillin treatment resulted into the significant reduction in the mitochondrial membrane depolarization and ROS production that eventually leads to apoptosis in HepG2 and SH-SY5Y cancer cells. These findings may offer a novel therapeutic approach by targeting the CAMKIV using natural product and its derivative with a minimal side effect.

Implications of molecular diversity of chitin and its derivatives.

Chitin is a long unbranched polysaccharide, made up of ß-1,4-linked N-acetylglucosamine which forms crystalline fiber-like structure. It is present in the fungal cell walls, insect and crustacean cuticles, nematode eggshells, and protozoa cyst. We provide a critical appraisal on the chemical modifications of chitin and its derivatives in the context of their improved efficacy in medical applications without any side effect. Recent advancement in nanobiotechnology has helped to synthesize several chitin derivatives having significant biological applications. Here, we discuss the molecular diversity of chitin and its applications in enzyme immobilization, wound healing, packaging material, controlled drug release, biomedical imaging, gene therapy, agriculture, biosensor, and cosmetics. Also, we highlighted chitin and its derivatives as an antioxidant, antimicrobial agent, anticoagulant material, food additive, and hypocholesterolemic agent. We envisage that chitin and chitosan-based nanomaterials with their potential applications would augment nanobiotechnology and biomedical industries.

From ZikV genome to vaccine: in silico approach for the epitope-based peptide vaccine against Zika virus envelope glycoprotein.

Zika virus (ZikV) has emerged as a potential threat to human health worldwide. A member of the Flaviviridae, ZikV is transmitted to humans by mosquitoes. It is related to other pathogenic vector-borne flaviviruses including dengue, West Nile and Japanese encephalitis viruses, but produces a comparatively mild disease in humans. As a result of its epidemic outbreak and the lack of potential medication, there is a need for improved vaccine/drugs. Computational techniques will provide further information about this virus. Comparative analysis of ZikV genomes should lead to the identification of the core characteristics that define a virus family, as well as its unique properties, while phylogenetic analysis will show the evolutionary relationships and provide clues about the protein's ancestry. Envelope glycoprotein of ZikV was obtained from a protein database and the most immunogenic epitope for T cells and B cells involved in cell-mediated immunity, whereas B cells are primarily responsible for humoral immunity. We mainly focused on MHC class I potential peptides. YRIMLSVHG, VLIFLSTAV and MMLELDPPF, GLDFSDLYY are the most potent peptides predicted as epitopes for CD4+ and CD8+ T cells, respectively, whereas MMLELDPPF and GLDFSDLYY had the highest pMHC-I immunogenicity score and these are further tested for interaction against the HLA molecules, using in silico docking techniques to verify the binding cleft epitope. However, this is an introductory approach to design an epitope-based peptide vaccine against ZikV; we hope that this model will be helpful in designing and predicting novel vaccine candidates.

Synthesis, Characterization, Antiglycation Evaluation, Molecular Docking, and ADMET Studies of 4-Thiazolidinone Derivatives.

The design and development of new small-molecule glycation inhibitors are essential for preventing various chronic diseases, including diabetes mellitus, immunoinflammation, cardiovascular, and neurodegenerative diseases. 4-Thiazolidinone or thiazolidine-4-one is a well-known heterocyclic compound with the potential to inhibit the formation of advanced glycation end products. In the present work, we report the synthesis and characterization of four new 5-arylidene 3-cyclopropyl-2-(phenylimino)thiazolidin-4-one (1-4) compounds and their human serum albumin glycation inhibitory activity. One of the compounds 5-(2H-1,3-benzodioxol-5-ylmethylidene)-3-cyclopropyl-2-(phenylimino)-1,3-thiazolidin-4-one (3) showed potent inhibition in the synthesis of initial, intermediary, and final products of glycation reactions. Besides, conformational changes in the α-helix and ß-sheet (due to hyperglycemia) were also found to be reversed upon the addition of (3). Experimental findings were complemented by computational [molecular docking, ADME/Tox, and density functional theory (DFT)] studies. The docking scores of the compounds were in order 1 > 3 > 2 > 4, indicating the importance of the polar group at the 5-arylidene moiety. The results of ADME/Tox and DFT calculations revealed the safe nature of the compounds with high drug-likeness and stability. Overall, we speculate that the results of this study could provide valuable insights into the biological activity of 4-thiazolidinones.

Identification of Novel Tau-Tubulin Kinase 2 Inhibitors Using Computational Approaches.

Tau tubulin kinase 2 (TTBK2) associated with multiple diseases is one of the kinases which phosphorylates tau and tubulin. Numerous efforts have been made to understand the role of TTBK2 in protein folding mechanisms and misfolding behavior. The misfolded protein intermediates form polymers with unwanted aggregation properties that initiate several diseases, including Alzheimer's. The availability of TTBK2 inhibitors can enhance the understanding of the molecular mechanism of action of the kinase and assist in developing novel therapeutics. In the quest for TTBK2 inhibitors, this study focuses on screening two chemical libraries (ChEMBL and ZINC-FDA). The molecular docking, RO5/absorption, distribution, metabolism, and excretion/toxicity, density functional theory, molecular dynamics (MD) simulations, and molecular mechanics with generalized Born and surface area solvation techniques enabled shortlisting of the four most active compounds, namely, ChEMBL1236395, ChEMBL2104398, ChEMBL3427435, and ZINC000000509440. Moreover, 500 ns MD simulation was performed for each complex, which provided valuable insights into the structural changes in the complexes. The relative fluctuation, solvent accessible surface area, atomic gyration, compactness covariance, and free energy landscapes revealed that the compounds could stabilize the TTBK2 protein. Overall, this study would be valuable for the researchers targeting the development of novel TTBK2 inhibitors.

Lead optimization, pharmacophore development and scaffold design of protein kinase CK2 inhibitors as potential COVID-19 therapeutics.

Therapeutic agents being designed against COVID-19 have targeted either the virus directly or the host cellular machinery. A particularly attractive host target is the ubiquitous and constitutively active serine-threonine kinase, Protein kinase CK2 (CK2). CK2 enhances viral protein synthesis by inhibiting the sequestration of host translational machinery as stress granules and assists in viral egression via association with the N-protein at filopodial protrusions of the infected cell. CK2 inhibitors such as Silmitasertib have been proposed as possible therapeutic candidates in COVID-19 infections. The present study aims to optimize Silmitasertib, develop pharmacophore models and design unique scaffolds to modulate CK2. The lead optimization phase involved the generation of compounds structurally similar to Silmitasertib via bioisostere replacement followed by a multi-stage docking approach to identify drug-like candidates. Molecular dynamics (MD) simulations were performed for two promising candidates (ZINC-43206125 and PC-57664175) to estimate their binding stability and interaction. Top scoring candidates from the lead optimization phase were utilized to build ligand-based pharmacophore models. These models were then merged with structure-based pharmacophores (e-pharmacophores) to build a hybrid hypothesis. This hybrid hypothesis was validated against a decoy set and used to screen a diverse kinase inhibitors library to identify favored chemical features in the retrieved actives. These chemical features include; an anion, an aromatic ring and an H-bond acceptor. Based on the knowledge of these features; de-novo scaffold design was carried out which identified phenindiones, carboxylated steroids, macrocycles and peptides as novel scaffolds with the potential to modulate CK2.Communicated by Ramaswamy H. Sarma.

In silico studies of alkaloids and their derivatives against N-acetyltransferase EIS protein from Mycobacterium tuberculosis.

Antibiotic resistance against Mycobacterium tuberculosis (M.tb.) has been a significant cause of death worldwide. The Enhanced intracellular survival (EIS) protein of the bacteria is an acetyltransferase that multiacetylates aminoglycoside antibiotics, preventing them from binding to the bacterial ribosome. To overcome the EIS-mediated antibiotics resistance of M.tb., we compiled 888 alkaloids and derivatives from five different databases and virtually screened them against the EIS receptor. The compound library was filtered down to 87 compounds, which underwent additional analysis and filtration. Moreover, the top 15 most prominent phytocompounds were obtained after the drug-likeness prediction and ADMET screening. Out of 15, nine compounds confirmed the maximum number of hydrogen bond interactions and reliable binding energies during molecular docking. Additionally, the Molecular dynamics (MD) simulation of nine compounds showed the three most stable complexes, further verified by re-docking with mutated protein. The density functional theory (DFT) calculation was performed to identify the HOMO-LUMO energy gaps of the selected three potential compounds. Finally, our selected top lead compounds i.e., Alkaloid AQC2 (PubChem85634496), Nobilisitine A (ChEbi68116), and N-methylcheilanthifoline (ChEbi140673) demonstrated more favourable outcomes when compared with reference compounds (i.e., 39b and 2i) in all parameters used in this study. Therefore, we anticipate that our findings will help to explore and develop natural compound therapy against multi and extensively drug-resistant strains of M.tb.Communicated by Ramaswamy H. Sarma.

In silico evaluation of 4-thiazolidinone-based inhibitors against the receptor for advanced glycation end products (RAGE).

Non-enzymatic glycation of biomolecules by reducing sugars led to several products, including the advanced glycation end products (AGEs), the accumulation of which has been linked to various life-threatening diseases. The binding of AGEs to their respective protein receptors for advanced glycation end products (RAGE) can initiate a cascade of reactions, which may alter physiological conditions. The present work investigates the potential of 4-thiazolidinones as RAGE inhibitors. We performed an extensive computational study to identify the structural requirements needed to act as RAGE inhibitors. To achieve this goal, 4-thiazolidinone-based compounds available in PubChem, ZINC15, ChEMBL, and ChEBI databases were screened against RAGE (PDB: 4LP5), leading to the identification of top five drug-like candidates with a high binding affinity to RAGE V-domain catalytic region. Drug likeness, absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the top-scoring compounds have been studied and discussed. Global molecular descriptors, chemical reactivity, hardness, softness, etc., have been estimated. Finally, molecular dynamics (MD) simulations at 100 ns were carried out to check the stability and other properties. Overall, we believe that the identified compounds can potentially attenuate RAGE-AGE interactions.Communicated by Ramaswamy H. Sarma.

Insights into the structural and dynamical changes of spike glycoprotein mutations associated with SARS-CoV-2 host receptor binding.

Novel Coronavirus or SARS-CoV-2 has received worldwide attention due to the COVID-19 pandemic, which originated in Wuhan, China leading to thousands of deaths to date. The SARS-CoV-2 Spike glycoprotein protein is one of the main focus of COVID-19 related research as it is a structural protein that facilitates its attachment, entry, and infection to the host cells. We have focused our work on mutations in two of the several functional domains in the virus spike glycoprotein, namely, receptor-binding domain (RBD) and heptad repeat 1 (HR1) domain. These domains are majorly responsible for the stability of spike glycoprotein and play a key role in the host cell attachment and infection. In our study, several mutations like R408I, L455Y, F486L, Q493N, Q498Y, N501T of RBD (319-591), and A930V, D936Y of HR1 (912-984) have been studied to examine its role on the spike glycoprotein native structure. Comparisons of MD simulations in the WT and mutants revealed a significant de-stabilization effect of the mutations on RBD and HR1 domains. We have investigated the impact of mapped mutations on the stability of the spike glycoprotein, before binding to the receptor, which may be consequential to its binding properties to the receptor and other ligands.Communicated by Ramaswamy H. Sarma.

Targeting SARS-CoV-2 nucleocapsid oligomerization: Insights from molecular docking and molecular dynamics simulations.

The outbreak of COVID-19 caused by SARS-CoV-2 virus continually led to infect a large population worldwide. Currently, there is no specific viral protein-targeted therapeutics. The Nucleocapsid (N) protein of the SARS-CoV-2 virus is necessary for viral RNA replication and transcription. The C-terminal domain of N protein (CTD) involves in the self-assembly of N protein into a filament that is packaged into new virions. In this study, the CTD (PDB ID: 6WJI) was targeted for the identification of possible inhibitors of oligomerization of N protein. Herein, multiple computational approaches were employed to explore the potential mechanisms of binding and inhibitor activity of five antiviral drugs toward CTD. The five anti-N drugs studied in this work are 4E1RCat, Silmitasertib, TMCB, Sapanisertib, and Rapamycin. Among the five drugs, 4E1RCat displayed highest binding affinity (-10.95 kcal/mol), followed by rapamycin (-8.91 kcal/mol), silmitasertib (-7.89 kcal/mol), TMCB (-7.05 kcal/mol), and sapanisertib (-6.14 kcal/mol). Subsequently, stability and dynamics of the protein-drug complex were examined with molecular dynamics (MD) simulations. Overall, drug binding increases the stability of the complex with maximum stability observed in the case of 4E1RCat. The CTD-drug complex systems behave differently in terms of the free energy landscape and showed differences in population distribution. Overall, the MD simulation parameters like RMSD, RMSF, Rg, hydrogen bonds analysis, PCA, FEL, and DCCM analysis indicated that 4E1RCat and TMCB complexes were more stable as compared to silmitasertib and sapanisertib and thus could act as effective drug compounds against CTD.Communicated by Ramaswamy H. Sarma.

Structural stability predictions and molecular dynamics simulations of RBD and HR1 mutations associated with SARS-CoV-2 spike glycoprotein.

The COVID-19 pandemic is caused by human transmission and infection of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2). There is no trusted drug against the virus; hence, efforts are on discovering novel inhibitors for the virus. The entry of a SARS-CoV-2 virus particle into a host cell is initiated by its spike glycoprotein and host Angiotensin-Converting Enzyme 2 (ACE2) receptor interaction. Spike glycoprotein domains, namely, the Receptor Binding Domain (RBD) and Heptad Repeat (HR) domains, are essential for this activity. We have studied the impact of mutations such as A348T, N354D, D364Y, G476S, V483A, S494D in the RBD (319-591), and S939F, S940T, T941A, S943P (912-984) in the HR1 domains of spike glycoprotein. Summarily, we utilized the computational screening algorithms to rank the deleterious, damaging and disease-associated spike glycoprotein mutations. Subsequently, to understand the changes in conformation, flexibility and function of the spike glycoprotein mutants, Molecular Dynamics (MD) simulations were performed. The computational predictions and analysis of the MD trajectories suggest that the RBD and HR1 mutations induce significant phenotypic effects on the pre-binding spike glycoprotein structure, which are presumably consequential to its binding to the receptor and provides lead to design inhibitors against the binding.Communicated by Ramaswamy H. Sarma.

Insights into the structure and dynamics of SARS-CoV-2 spike glycoprotein double mutant L452R-E484Q.

The Receptor Binding Domain (RBD) of SARS-CoV-2, located on the S1 subunit, plays a vital role in the virus binding and its entry into the host cell through angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, understanding the dynamic effects of mutants on the SARS-CoV-2 RBD is essential for discovering drugs to inhibit the virus binding and disrupt its entry into the host cells. A recent study reported a double mutant of SARS-CoV-2, L452R-E484Q, located in the RBD region. Thus, this study employed various computational algorithms and methods to understand the structural impact of both individual variants L452R, E484Q, and the double mutant L452R-E484Q on the native RBD of spike glycoprotein. The effects of the mutations on native RBD structure were predicted by in silico algorithms, which predicted changes in the protein structure and function upon the mutations. Subsequently, molecular dynamics (MD) simulations were employed to understand the conformational stability and functional changes on the RBD upon the mutations. The comparative results of MD simulation parameters displayed that the double mutant induces significant conformational changes in the spike glycoprotein RBD, which may alter its biological functions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03151-0.

Anti-Fungal Drug Anidulafungin Inhibits SARS-CoV-2 Spike-Induced Syncytia Formation by Targeting ACE2-Spike Protein Interaction.

Drug repositioning continues to be the most effective, practicable possibility to treat COVID-19 patients. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters target cells by binding to the ACE2 receptor via its spike (S) glycoprotein. We used molecular docking-based virtual screening approaches to categorize potential antagonists, halting ACE2-spike interactions by utilizing 450 FDA-approved chemical compounds. Three drug candidates (i.e., anidulafungin, lopinavir, and indinavir) were selected, which show high binding affinity toward the ACE2 receptor. The conformational stability of selected docked complexes was analyzed through molecular dynamics (MD) simulations. The MD simulation trajectories were assessed and monitored for ACE2 deviation, residue fluctuation, the radius of gyration, solvent accessible surface area, and free energy landscapes. The inhibitory activities of the selected compounds were eventually tested in-vitro using Vero and HEK-ACE2 cells. Interestingly, besides inhibiting SARS-CoV-2 S glycoprotein induced syncytia formation, anidulafungin and lopinavir also blocked S-pseudotyped particle entry into target cells. Altogether, anidulafungin and lopinavir are ranked the most effective among all the tested drugs against ACE2 receptor-S glycoprotein interaction. Based on these findings, we propose that anidulafungin is a novel potential drug targeting ACE2, which warrants further investigation for COVID-19 treatment.

Strand 1A variant in neuroserpin shows increased aggregation and no loss of inhibition: implication in ameliorating polymerization to retain activity.

Neuroserpin (NS) is predominantly expressed in the brain and is the primary inhibitor of tissue plasminogen activator (tPA). NS variants are associated with the neurogenerative disease termed familial encephalopathy with neuroserpin inclusion bodies (FENIB). The disease is characterized by variable age of onset and severity. The reactive center loop (RCL) insertion-based inhibitory mechanism of NS requires a coordinated conformational change leading to a shift in the strands of the ß-sheet A and movement of helix F. Strand 1A is connected to the helix F at its C terminal end and with the strand 2A at its N terminal, both these domain move for accommodating the inserting loop; therefore, a variant that influences their movement may alter the inhibition rates. A molecular dynamic simulation analysis of a H138C NS variant from strand 1A showed a large decrease in conformational fluctuations as compared with wild-type NS. H138 was mutated, expressed, purified and a native-PAGE and transmission electron microscopy (TEM) analysis showed that this variant forms large molecular weight aggregates on a slight increase in temperature. However, a circular dichroism analysis showed its secondary structure to be largely conserved. Surprisingly, its tPA inhibition activity and complex formation remain unhindered even after the site-specific labeling of H138C with Alexa fluor C5 maleimide. Further, a helix F-strand 1A (W154C-H138C) double variant still shows appreciable inhibitory activity. Increasingly, it appears that aggregation and not loss of inhibition is the more likely cause of shutter region-based variants phenotypes, indicating that hindering polymer formation using small molecules may retain inhibitory activity in pathological variants of NS.

Identification of PARP12 Inhibitors By Virtual Screening and Molecular Dynamics Simulations.

Poly [adenosine diphosphate (ADP)-ribose] polymerases (PARPs) are members of a family of 17 enzymes that performs several fundamental cellular processes. Aberrant activity (mutation) in PARP12 has been linked to various diseases including inflammation, cardiovascular disease, and cancer. Herein, a large library of compounds (ZINC-FDA database) has been screened virtually to identify potential PARP12 inhibitor(s). The best compounds were selected on the basis of binding affinity scores and poses. Molecular dynamics (MD) simulation and binding free energy calculation (MMGBSA) were carried out to delineate the stability and dynamics of the resulting complexes. To this end, root means deviations, relative fluctuation, atomic gyration, compactness, covariance, residue-residue contact map, and free energy landscapes were studied. These studies have revealed that compounds ZINC03830332, ZINC03830554, and ZINC03831186 are promising agents against mutated PARP12.