Mining therapeutic targets from the antibiotic-resistant Campylobacter coli and virtual screening of natural product inhibitors against its riboflavin synthase.

Campylobacter coli resides in the intestine of several commonly consumed animals, as well as water and soil. It leads to campylobacteriosis when humans eat raw/undercooked meat or come into contact with infected animals. A common manifestation of the infection is fever, nausea, headache, and diarrhea. Increasing antibiotic resistance is being observed in this pathogen. The increased incidence of C. coli infection, and post-infection complications like Guillain-Barré syndrome, make it an important pathogen. It is essential to find novel therapeutic targets and drugs against it, especially with the emergence of antibiotic-resistant strains. In the current study, genomes of 89 antibiotic-resistant strains of C. coli were downloaded from the PATRIC database. Potent drug targets (n = 36) were prioritized from the core genome (n = 1,337 genes) of this species. Riboflavin synthase was selected as a drug target and pharmacophore-based virtual screening was performed to predict its inhibitors from the NPASS (n = ~ 30,000 compounds) natural product library. The top three docked compounds (NPC115144, NPC307895, and NPC470462) were selected for dynamics simulation (for 50 ns) and ADMET profiling. These identified compounds appear safe for targeting this pathogen and can be further validated by experimental analysis before clinical trials.

An integrated in silico based subtractive genomics and reverse vaccinology approach for the identification of novel vaccine candidate and chimeric vaccine against XDR Salmonella typhi H58.

Salmonella typhi is notorious for causing enteric fever which is also known as typhoid fever. It emerged as an extreme drug resistant strain that requires urgent attention to prevent its global spread. Statistically, about 11-17 million typhoid illnesses are reported worldwide annually. The only alternative approach for the control of this illness is proper vaccination. However, available typhoid vaccine has certain limitations such as poor long-term efficacy, and non-recommendation for below 6 years children, which opens the avenues for designing new vaccines to overcome such limitations. Computational-based reverse vaccinology along with subtractive genomics analysis is one of the robust approaches used for the prioritization of vaccine candidates through direct screening of genome sequence assemblies. In the current study, we have successfully designed a peptide-based novel antigen chimeric vaccine candidate against the XDR strain of S. typhi H58. The pipeline revealed four peptides from WP_001176621.1 i.e., peptidoglycan-associated lipoprotein Pal and two peptides from WP_000747548.1 i.e., OmpA family lipoprotein as promising target for the induction of immune response against S. typhi. The six epitopes from both proteins were found as immunogenic, antigenic, virulent, highly conserved, nontoxic, and non-allergenic among whole Salmonella H58 proteome. Furthermore, the binding interaction between a chimeric vaccine and human population alleles was unveiled through structure-based studies. So far, these proteins have never been characterized as vaccine targets against S. typhi. The current study proposed that construct V2 could be a significant vaccine candidate against S. typhi H58. However, to ascertain this, future experimental holistic studies are recommended as follow-up.

Selection of Multi-Drug Targets against Drug-Resistant Mycobacterium tuberculosis XDR1219 Using the Hyperbolic Mapping of the Protein Interaction Network.

Tuberculosis remains the leading cause of death from a single pathogen. On the other hand, antimicrobial resistance (AMR) makes it increasingly difficult to deal with this disease. We present the hyperbolic embedding of the Mycobacterium tuberculosis protein interaction network (mtbPIN) of resistant strain (MTB XDR1219) to determine the biological relevance of its latent geometry. In this hypermap, proteins with similar interacting partners occupy close positions. An analysis of the hypermap of available drug targets (DTs) and their direct and intermediate interactors was used to identify potentially useful drug combinations and drug targets. We identify rpsA and rpsL as close DTs targeted by different drugs (pyrazinamide and aminoglycosides, respectively) and propose that the combination of these drugs could have a synergistic effect. We also used the hypermap to explain the effects of drugs that affect multiple DTs, for example, forcing the bacteria to deal with multiple stresses like ethambutol, which affects the synthesis of both arabinogalactan and lipoarabinomannan. Our strategy uncovers novel potential DTs, such as dprE1 and dnaK proteins, which interact with two close DT pairs: arabinosyltransferases (embC and embB), Ser/Thr protein kinase (pknB) and RNA polymerase (rpoB), respectively. Our approach provides mechanistic explanations for existing drugs and suggests new DTs. This strategy can also be applied to the study of other resistant strains.

Construction of a comprehensive protein-protein interaction map for multi drug resistant klebsiella pneumoniae to identify drug targets: A computational approach.

Klebsiella pneumoniae is an encapsulated rod-shaped, Gram-negative microbe that can form biofilm. It is an opportunistic Enterobacter usually involved in nosocomial infection, conferring resistance to almost all antibiotics and hence become therapeutically challenging. In the current study, the Protein Interaction Network (PIN) of MDR K. pneumoniae has been identified. The proteins are the building blocks of all organisms. Proteins interact with each other to carry out their physiological functions. The interactions are integrated to form Protein Interaction Network (PIN). The strain DA48896 has been selected as it was isolated from Pakistan and harboring bla-oxa-181, conferring resistance to carbapenem. Total 20,936 high confidence interactions of 3782 proteins have been predicted from the STRING database. The predicted interactions were annotated functionally and mapped on their corresponding pathways. The predicted PIN was verified using semantic similarity between the Gene Ontology. The topological properties were calculated and retrieved topologically significant proteins consisting of 390 proteins. Among them 49 proteins are non-homologous essential that can serve as the potential drug targets. These proteins were further explored for druggability, their association with pathways involved in drug resistance and eventually prioritized as potential drug targets. This study will be helpful to design drug candidates against prioritized proteins.

Integrated bioinformatics based subtractive genomics approach to decipher the therapeutic function of hypothetical proteins from Salmonella typhi XDR H-58 strain.

PURPOSE: The efficacy of drugs against Salmonella infection have compromised due to emerging XDR H58 strain. There is a dire need to find novel antimicrobial drug targets as well as drug candidates to cure by the XDR strain of Salmonella. It is observed that the complete genome sequence of the XDR H58 strain contains a large number of hypothetical proteins with unknown cellular and biological functions. Hence, it is indispensable to annotate these proteins functionally as well as structurally to identify novel drug targets. METHODS: In the current study, a comparative genomics and proteomics based approach was applied to find the novel drug targets in XDR strain while comparing the MDR and NR strains of Salmonella typhi. RESULTS: The characterization of ~ 350 hypothetical proteins were performed through determination of their physio-chemical properties, sub-cellular localization, functional annotation, and structure-based studies. As a result, only five proteins were prioritized as essential, druggable, and virulent proteins. Moreover, only one protein i.e. WP_000916613.1 was functionally annotated with high confidence and subjected to further structure-based analysis. CONCLUSION: The current study presents a hypothetical protein from the XDR S. typhi proteome as a potential pharmacological target against which novel therapeutic candidates may be predicted. The outcome of the current study may lead to formulate a general set of pipelines for better understanding of the role of hypothetical proteins in pathogenesis of not only Salmonella but also for other pathogens.

Total Bioactive Contents, Metabolic Profiling, Docking Studies, Antioxidant and Enzyme Inhibition Activities of Convolvulus Arvensis L. and Multivariate Analysis to Unravel a Potential Herb as Natural Resource for Pharmaceutical Industry.

Convolvulus arvensis L. is an evergreen herb growing in various regions of Pakistan. Despite of several medicinal properties associated to this herb, it was not investigated scientifically for its bioactive compounds and detailed pharmaceutical properties. Therefore, its methanolic extract was divided into hexane (CA-H), chloroform (CA-C), ethyl acetate (CA-E) and butanol (CA-B) soluble fractions. CA-H and CA-C were found rich in phenolics (30.73±0.63 and 20.15±0.59 mg GAE/g of the extract, respectively), and the same fractions exhibited significant antioxidant activities (DPPH: 5.23±0.11 & 12.34±0.17 mg TE/g extract, respectively; ABTS: 36.82±0.04 & 56.74±0.61 mg TE/g extract, respectively). Also in CUPRAC activity assay, CA-H and CA-C exhibited highest activities as 87.30±0.46 and 56.74±0.61 mg TE/g extract, respectively, while CA-C was most active in FRAP activity assay with value of 40.21±2.19 mg TE/g extract. Total antioxidant capacity (1.23±0.033 mmol TE/g extract) was also found higher for CA-C, while CA-H activity was also comparable, however, CA-H showed higher metal chelating activity (22.74±0.001 mg EDTAE/g extract) than that of CA-C (17.55±0.22 mg EDTAE/g extract). These activities clearly revealed a direct relation between antioxidant potential and phenolic contents of CA-H and CA-C. In AChE and BChE inhibitory assay, CA-H and CA-E showed better inhibition (AChE: 8.24±0.77 & 4.46±0.007 mg GALAE/g extract; BChE: 5.40±0.02 & 1.92±0.24 mg GALAE/g extract) as compared to other fractions, whereas, against tyrosinase, CA-B was most active (37.35±0.53 mg KAE/g extract). CA-H and CA-C also showed higher inhibitory potential (0.98±0.08 & 0.58±0.01 mmol ACAE/g extract) against α-Amylase; while against α-Glucosidase, CA-E was the most active fraction. UHPLC/MS analysis of the methanolic extract of C. arvensis disclosed the presence of 62 compounds as sterols, triterpenes, flavonoids, fatty acids, alkaloids and coumarins. In Multivariate Analysis, the total phenolic contents were correlated strongly with all antioxidant assays except FRAP and DPPH. Regarding enzyme inhibitory properties, only AChE, BChE and α-amylase were correlated with the total phenolic contents in the extracts. Docking analyses confirmed these findings, as identified compounds had high binding free energy and inhibition constants with the enzymes studied. It was finally concluded that C. arvensis is a potential industrial crop, which can be a component of nutraceuticals and functional foods, if evaluated for its toxicity.

Re-purposing of hepatitis C virus FDA approved direct acting antivirals as potential SARS-CoV-2 protease inhibitors.

A new coronavirus strain called as SARS-CoV-2 has emerged from Wuhan, China in late 2019 and it caused a worldwide pandemic in a few months. After the Second World War, it is the biggest calamity observed as there is no specific US Food and Drugs Administration (USFDA) approved drug or vaccine available globally for the treatment. Several clinical trials are ongoing for therapeutic alternatives, however with little success rate. Considering that the time is crucial, the drug repurposing and data obtained from in silico models are one of the most important approaches to identify possible lead inhibitors against SARS-CoV-2. More recently, the Direct Acting Antivirals (DAAs) are emerged as the most promising drugs to control viral infection. The Main Protease (Mpro), a key enzyme in the SARS-CoV-2 replication cycle, is found close homolog to the Hepatitis C Virus (HCV) protease and could be susceptible of blocking its activity by DAAs. In the current study, the DAAs were investigated as antivirals using structure based computational approach against Mpro of SARS-CoV-2 to propose them as new therapeutics. In total, 20 DAAs of HCV, including a reference compound O6K were docked against Mpro. The docked structures were examined and resulted in the identification of six highly promising DAAs i.e. beclabuvir, elbasvir, paritaprevir, grazoprevir, simeprevir, and asunapevir exhibiting high theoretical binding affinity to Mpro from SARS-CoV-2 in comparison to other DAAs. Furthermore, the post docking analysis revealed that Cys145, Glu166, His163, Thr26, His41, and Met165 played potential role for the binding of these DAAs inside binding site of Mpro. Furthermore, the correlation between binding energies were found in accord with the results from the reported IC50s for some DAAs. Overall, the current study provides insight to combat COVID-19 using FDA-approved DAAs as repurposed drugs.

A UPLC-DAD-Based Bio-Screening Assay for the Evaluation of the Angiotensin Converting Enzyme Inhibitory Potential of Plant Extracts and Compounds: Pyrroquinazoline Alkaloids from Adhatoda vasica as a Case Study.

Angiotensin converting enzyme (ACE) plays a crucial role in regulating blood pressure in the human body. Identification of potential ACE inhibitors from medicinal plants supported the idea of repurposing these medicinal plants against hypertension. A method based on ultra-performance liquid chromatography (UPLC) coupled with a diode array detector (DAD) was used for the rapid screening of plant extracts and purified compounds to determine their ACE inhibitory activity. Hippuryl-histidiyl-leucine (HHL) was used as a substrate, which is converted into hippuric acid (HA) by the action of ACE. A calibration curve of the substrate HHL was developed with the linear regression 0.999. The limits of detection and quantification of this method were found to be 0.134 and 0.4061 mM, respectively. Different parameters of ACE inhibitory assay were optimized, including concentration, incubation time and temperature. The ACE inhibition potential of Adhatoda vasica (methanolic-aqueous extract) and its isolated pyrroquinazoline alkaloids, vasicinol (1), vasicine (2) and vasicinone (3) was evaluated. Compounds 1-3 were characterized by various spectroscopic techniques. The IC50 values of vasicinol (1), vasicine (2) and vasicinone (3) were found to be 6.45, 2.60 and 13.49 mM, respectively. Molecular docking studies of compounds 1-3 were also performed. Among these compounds, vasicinol (1) binds as effectively as captopril, a standard drug of ACE inhibition.

Comparative proteome-wide study for in-silico identification and characterization of indispensable hypothetical proteins of food borne-pathogen Campylobacter jejuni (CJJ) by subtractive genomics approach.

Campylobacter jejuni (CJJ) is a source of bacterial foodborne diarrhea globally. Mostly found prevalent in children in the developing countries that may lead to mortality. The upsurge in antimicrobial resistance is causing hindrance in the treatment, as highlighted by CDC and WHO. The study hypothesized the application of subtractive genomics approach coupled with metabolic pathway to reveal unidentified essential proteins that could serve as potential drug target (s). The approach was employed to model the druggable proteome of C. jejuni resistant strain 81-176. We obtained 728/1744 non-homologous essential proteins by performing sequence similarity search against host proteome and DEG server, respectively. The KAAS annotated metabolic pathway information; PSORTb predicted their sub cellular localization and SVMPro functional annotated 104 hypothetical proteins while the Drug Bank for the druggability analysis. We found 04/104 protein druggable viz. synaptic vesicular amine transporter, Uracil-DNA glycosylase, Laccase domain protein YfiH, and Phosphoenolpyruvate protein phosphor transferase. The study has revealed a formerly uncharacterized pool of C. jejuni proteins that can play a significant role in controlling CJJ infection and presented previously uncharacterized four proteins as potential drug targets. These potential drug targets can further be explored employing structure-based and other biochemical methods by the scientific community.

A computational subtractive genome analysis for the characterization of novel drug targets in Klebsiella pneumonia strain PittNDM01.

The emergence of carbapenem-resistant Klebsiella Pneumoniae had been reported previously, which needs rapid attention. Currently, Pittsburgh University Hospital reported a new strain of carbapenem-resistant Klebsiella pneumoniae that was co-producing OXA-232 and NDM-1 named as PittNDM01. This strain is resistant to almost all beta-lactam antibiotics such as Carbapenem as well as to fluoroquinolones and aminoglycosides. Globally, failure to the wide-spread pathogenic strains had been observed due to the increased and antibiotic resistance, which leads to less antimicrobial drug efficacy. Since last decades, computational genomic approaches have been introduced to fight against resistant pathogens, which is an advanced approach for novel drug targets investigation. The current study emphasizes the utilization of the available genomic and proteomic data of Klebsiella pneumoniae PittNDM01 for the identification of novel drug targets for future drug developments. Comparative genomic analysis and molecular biological tools were applied, results in observing 582 non-human homologous-essential proteins of Klebsiella pneumoniae. Among the total 582 proteins, 66 were closely related to the pathogen-specific pathway. Out of all 66-targeted proteins, ten non-homologous essential proteins were found to have druggability potential. The subcellular localization of these proteins revealed; 6 proteins in the cytoplasm, 2 in the inner membrane, and one each in periplasmic space and outer membrane. All the above 10 proteins were compared to the proteins sequences of gut flora to eliminate the homologous proteins. In total, 6-novel non-human and non-gut flora essential drug targets of Klebsiella pneumoniae PittNDM01 strain were identified. Further, the 3D structures of the identified drug target proteins were developed, and protein-protein interaction network analysis was performed to know the functional annotation of the desire proteins. Therefore, these non-homologous essential targets ensure the survival of the pathogen and hence can be targeted for drug discovery.

Identification of putative non-host essential genes and novel drug targets against Acinetobacter baumannii by in silico comparative genome analysis.

Acinetobacter baumannii, the gram-negative bacteria emerged as an extremely critical pathogen causing nosocomial and different kinds of infections. A. baumannii exhibit resistivity towards various classes of antibiotics that shows that there is a dire need to search more drug targets by exploiting the full genome of the bacteria. In doing so, a strategy is made with the combination of computational biology, pathogen informatics and cheminformatics. Comparative genomics analysis, modeling and docking studies have been performed for the prediction of non-host essential genes and novel drug candidates against A. baumannii. Among 37 unique and 82 common metabolic pathways, 92 genes were predicted as non-host genes. Similarly, using homology search between A. baumannii genome and essential genes of different bacteria, 293 genes were predicted as essential genes of A. baumannii. Among these predicted non-host and essential genes, 86 genes were predicted as non-host essential genes which could serve as potential novel drug and vaccine targets. Additional drug-target like physicochemical properties were estimated such as the molecular weight, subcellular localization and druggability potential. On the structural part, the crystal structures of all the non-host essential genes of A. baumannii were found except the three genes. Out of these three, a homology model of Undecaprenyl-diphosphatase was built using a PDB template by MODELLER [version 9.18]. The quality of the model was assessed by the ProSA and RAMPAGE. The built model was subjected as a receptor for the molecular docking with Adenosine diphosphate (ADP) as a ligand. The molecular docking was performed by AutoDock4 and the best conformation with lowest binding energy (-4.39 kcal/mol) was obtained. The LigPlot was used to identify the close interactions between the ligand the receptor's residues. This study will further aid for the selection of putative inhibitors against a novel drug target identified against A. baumannii and hence could lead to the better therapeutics.

Subtractive genome analysis for in silico identification and characterization of novel drug targets in Streptococcus pneumonia strain JJA.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive bacterium. Humans are the major target for the pneumococcus. The pneumococcus is a common etiological agent of many different diseases such as bacterial meningitis, pneumonia, otitis media (OM), sinusitis, and conjunctivitis. According to the WHO, the pneumococcus is responsible for causing 1 million deaths each year. In 2000, over 14 million children worldwide under the age of 5 years were diagnosed with a pneumococcal disease, with the highest incidence seen in Africa. The human population most susceptible to pneumococcal infections is that of children due to their immature immune system. A sensational increase in antibiotic resistance among S. pneumoniae has been witnessed in different parts of the world since 1980s. The increase of resistance of S. pneumoniae to antibiotics is of major concern throughout the world. Worldwide, there are concerns about rising levels of antibiotic resistance and fears that the efficacy of antimicrobial therapy may be compromised, resulting in treatment failure and reduced utility of older antibiotics, a comparatively novel method has been used to defeat the resistant pathogens since last decade. The computational subtractive genomics approach is one of them, in which the bacterial pathogen complete proteins is gradually rock-bottom to a small number of likely drug targets. In this approach the steps which are used to find human non-homologs targets, proteins that are essential to the disease causing agent and participation of the selected proteins in pathogen metabolic pathways which are necessary for the survival of bacteria. We used computational subtractive genomics on consummate proteins of the of S. pneumonia strain JJA in this study and concluded with 2 proteins that can be used as potent drug targets against which new dynamic molecules can be planned to make better the action to treat the disease which is related with pathogen.

Proteome-wide identification of epitope-based vaccine candidates against multi-drug resistant Proteus mirabilis.

Proteus mirabilis is one of the important pathogens of urinary tract and exhibits resistance to multiple drugs. Development of vaccine tends to be the most promising and cost-effective remedy against the said pathogen. Herein, we implement a combinatorial approach for screening proteins harboring potential broad-spectrum antigenic epitopes in the proteome of P. mirabilis. The targets are host non-homologous, essential and virulent, and have localization in the extracellular and outer membrane. Immuno-informatics revealed antigenic, surface exposed and broad-spectrum B-cell derived T-cell epitopes for three membrane usher family candidates: AtfC, PMI2533 and PMI1466, which could evoke a substantial immune response. Protein-protein interactions of targeted three proteins have shown their involvement in biologically significant pathways indispensable for the growth and survival of the pathogen. The antigenic epitopes are conserved among all completely annotated strains and docked deeply in the binding cavity of the most prevalent allele-DRB1*0101 in human population. Future work is necessary to characterize the shortlisted proteins and epitopes for immune protection in animal models.

Synthesis of 4-thiazolidinone analogs as potent in vitro anti-urease agents.

4-Thiazolidinone analogs 1-20 were synthesized, characterized by (1)H NMR and EI-MS and investigated for urease inhibitory activity. All twenty (20) analogs exhibited varied degree of urease inhibitory potential with IC50 values 1.73-69.65µM, if compared with standard thiourea having IC50 value of 21.25±0.15µM. Among the series, eight derivatives 3, 6, 8, 10, 15, 17, 19, and 20 showed outstanding urease inhibitory potential with IC50 values of 9.34±0.02, 14.62±0.03, 8.43±0.01, 7.3±0.04, 2.31±0.002, 5.75±0.003, 8.81±0.005, and 1.73±0.001µM, respectively, which is better than the standard thiourea. The remaining analogs showed good to excellent urease inhibition. The binding interactions of these compounds were confirmed through molecular docking studies.

An exhaustive yet simple virtual screening campaign against Sortase A from multiple drug resistant Staphylococcus aureus.

Methicillin resistant Staphylococcus aureus (MRSA) is one of the challenging bacterial pathogen due to its acquired resistance to the ß lactam antibiotics. The Sortase A is an enzyme of Gram-positive bacteria including S. aureus to anchor surface proteins to the cell wall. Sortase A is well studied enzyme and considered as the drug target against MRSA. Sortase A plays active role in anchoring the virulence proteins on the cell wall of the Gram-positive bacteria. The inhibition of Sortase A activity results in the separation of S. aureus from the host cells and ultimately alleviation of the infection. Here, we adapted a structure-based virtual screening protocol which helped in identification of novel potential inhibitors of Sortase A. The protocol involved the docking of a chemical library of druglike compounds with the Sortase A binding site represented by multiple crystal structures. The compounds were ranked by multiple scoring functions and shortlisted for future experimental screening. The method resulted in shortlisting of three compounds as potential novel inhibitors of Sortase A out of a large chemical library. The high rankings of shortlisted compounds estimated by multiple scoring functions showed their binding potential with Sortase A. The results are proved to be a simple yet efficient choice of structure-based virtual screening. The identified compounds are druglike and show high rankings among all set protocols of the virtual screening. We hope that the study would eventually help to expedite the discovery of novel drug candidates against MRSA.

Integrating core subtractive proteomics and reverse vaccinology for multi-epitope vaccine design against Rickettsia prowazekii endemic typhus.

Rickettsia prowazekii is an intracellular, obligate, gram-negative coccobacillus responsible for epidemic typhus. Usually, the infected body louse or its excrement when rubbed into the skin abrasions transmits the disease. The infection with R. prowazekii causes the highest death rate (> 20% without antibiotic treatment and now 1-7%), followed by epidemic typhus, which often manifests in unsanitary conditions (up to 15-30%). Conventionally, vaccine design has required pathogen growth and both assays (in vivo and in vitro), which are costly and time-consuming. However, advancements in bioinformatics and computational biology have accelerated the development of effective vaccine designs, reducing the need for traditional, time-consuming laboratory experiments. Subtractive genomics and reverse vaccinology have become prominent computational methods for vaccine model construction. Therefore, the RefSeq sequence of Rickettsia prowazekii (strain Madrid E) (Proteome ID: UP000002480) was subjected to subtractive genomic analysis, including factors such as non-similarity to host proteome, essentiality, subcellular localization, antigenicity, non-allergenicity, and stability. Based on these parameters, the vaccine design process selected specific proteins such as outer membrane protein R (O05971_RICPR PETR; OmpR). Eventually, the OmpR was subjected to a reverse vaccinology approach that included molecular docking, immunological simulation, and the discovery of B-cell epitopes and MHC-I and MHC-II epitopes. Consequently, a chimeric or multi-epitope-based vaccine was proposed by selecting the V11 vaccine and its 3D structure modeling along with molecular docking against TLR and HLA protein, in silico simulation, and vector designing. The obtained results from this investigation resulted in a new perception of inhibitory ways against Rickettsia prowazekii by instigating novel immunogenic targets. To further assess the efficacy and protective ability of the newly designed V11 vaccine against Rickettsia prowazekii infections, additional evaluation such as in vitro or in vivo immunoassays is recommended.

Endogenous tau released from human ReNCell VM cultures by neuronal activity is phosphorylated at multiple sites.

Tau is an intracellular protein but also known to be released into the extracellular fluid. Tau release mechanisms have drawn intense attention as these are known to play a key role in Alzheimer's disease (AD) pathology. However, tau can also be released under physiological conditions although its physiological function and release mechanisms have been poorly characterized, especially in human neuronal cells. We investigated endogenous tau release in ReNCell VM, a human neuroprogenitor cell line, under physiological conditions and found that tau is spontaneously released from cells. To study activity-dependent release of endogenous tau, human ReNCell VM culture was stimulated by 100µM AMPA or 50mM KCl for one-hour, tau was actively released to the culture medium. The released tau was highly phosphorylated at nine phosphorylation sites (pSites) detected by phospho-specific tau antibodies including AT270 (T175/T181), AT8 (S202/T205), AT100 (T212/S214), AT180 (T231), and PHF-1 (S396/S404), showing that these pSites are important for activity-dependent tau release from human ReNCell VM. Intracellular tau showed various phosphorylation status across these sites, with AT270 and PHF-1 highly phosphorylated while AT8 and AT180 were minimally phosphorylated, suggesting that AT8 and AT180 pSites exhibit a propensity for secretion rather than being retained intracellularly. This activity-dependent tau release was significantly decreased by inhibition of GSK-3ß, demonstrating that GSK3ß-dependent phosphorylation of tau plays an important role in its release by neuronal activity. In this study, we showed that ReNCell VM serves as a valuable model for studying endogenous physiological tau release. Further, ReNCell model can be also used to study pathological release of human tau that will contribute to our understanding of the progression of AD and related dementias.

Identification of therapeutic drug target of Shigella Flexneri serotype X through subtractive genomic approach and in-silico screening based on drug repurposing.

Shigellosis, induced by Shigella flexneri, constitutes a significant health burden in developing nations, particularly impacting socioeconomically disadvantaged communities. Designated as the second most prevalent cause of diarrheal illness by the World Health Organization (WHO), it precipitates an estimated 212,000 fatalities annually. Within the spectrum of S. flexneri strains, serotype X is notably pervasive and resilient, yet its comprehensive characterization remains deficient. The present investigation endeavors to discern potential pharmacological targets and repurpose existing drug compounds against S. flexneri serotype X. Employing the framework of subtractive genomics, the study interrogates the reference genome of S. flexneri Serotype X (strain 2,002,017; UP000001884) to delineate its proteome into categories of non-homologous, non-paralogous, essential, virulent, and resistant constituents, thereby facilitating the identification of therapeutic targets. Subsequently, a screening of approximately 9000 compounds from the FDA library against the identified drug target aims to delineate efficacious agents for combating S. flexneri serotype X infections. The application of subtractive genomics methodology yields prognostic insights, unveiling non-paralogous proteins (n = 4122), non-homologues (n = 1803), essential (n = 1246), drug-like (n = 389), resistant (n = 167), alongside 42 virulent proteins within the reference proteome. This iterative process culminates in the identification of Serine O-acetyltransferase as a viable drug target. Subsequent virtual screening endeavors to unearth FDA-approved medicinal compounds capable of inhibiting Serine O-acetyltransferase. Noteworthy candidates such as DB12983, DB15085, DB16098, DB16185, and DB16262 emerge, exhibiting potential for mitigating S. flexneri Serotype X. Despite the auspicious findings, diligent scrutiny is imperative to ascertain the efficacy and safety profile of the proposed drug candidates vis-à-vis S. flexneri.

Immunoinformatic-guided designing of multi-epitope vaccine construct against Brucella Suis 1300.

Brucella suis mediates the transmission of brucellosis in humans and animals and a significant facultative zoonotic pathogen found in livestock. It has the capacity to survive and multiply in a phagocytic environment and to acquire resistance under hostile conditions thus becoming a threat globally. Antibiotic resistance is posing a substantial public health threat, hence there is an unmet and urgent clinical need for immune-based non-antibiotic methods to treat brucellosis. Hence, we aimed to explore the whole proteome of Brucella suis to predict antigenic proteins as a vaccine target and designed a novel chimeric vaccine (multi-epitope vaccine) through subtractive genomics-based reverse vaccinology approaches. The applied subsequent hierarchical shortlisting resulted in the identification of Multidrug efflux Resistance-nodulation-division (RND) transporter outer membrane subunit (gene BepC) that may act as a potential vaccine target. T-cell and B-cell epitopes have been predicted from target proteins using a number of immunoinformatic methods. Six MHC I, ten MHC II, and four B-cell epitopes were used to create a 324-amino-acid MEV construct, which was coupled with appropriate linkers and adjuvant. To boost the immunological response to the vaccine, the vaccine was combined with the TLR4 agonist HBHA protein. The MEV structure predicted was found to be highly antigenic, non-toxic, non-allergenic, flexible, stable, and soluble. To confirm the interactions with the receptors, a molecular docking simulation of the MEV was done using the human TLR4 (toll-like receptor 4) and HLAs. The stability and binding of the MEV-docked complexes with TLR4 were assessed using molecular dynamics (MD) simulation. Finally, MEV was reverse translated, its cDNA structure was evaluated, and then, in silico cloning into an E. coli expression host was conducted to promote maximum vaccine protein production with appropriate post-translational modifications. These comprehensive computer calculations backed up the efficacy of the suggested MEV in protecting against B. suis infections. However, more experimental validations are needed to adequately assess the vaccine candidate's potential. HIGHLIGHTS: • Subtractive genomic analysis and reverse vaccinology for the prioritization of novel vaccine target • Examination of chimeric vaccine in terms of allergenicity, antigenicity, MHC I, II binding efficacy, and structural-based studies • Molecular docking simulation method to rank based vaccine candidate and understand their binding modes.

Pangenome profiling of novel drug target against vancomycin-resistant Enterococcus faecium.

Enterococcus faecium is a frequent causative agent of nosocomial infection mainly acquired from outgoing hospital patients (Hospital Acquired Infection-HAIs). They are largely involved in the outbreaks of bacteremia, UTI, and endocarditis with a high transmissibility rate. The recent emergence of VRE strain (i.e. vancomycin resistant enterococcus) turned it into high priority pathogen for which new drug research is of dire need. Therefore, in current study, pangenome and resistome analyses were performed for available antibiotic-resistant genomes (n = 216) of E. faecium. It resulted in the prediction of around 5,059 genes as an accessory gene, 1,076 genes as core and 1,558 genes made up a unique genome fraction. Core genes common to all strains were further used for the identification of potent drug targets by applying subtractive genomics approach. Moreover, the COG functional analysis showed that these genomes are highly enriched in metabolic pathways such as in translational, ribosomal, proteins, carbohydrates and nucleotide transport metabolism. Through subtractive genomics it was observed that 431 proteins were non-homologous to the human proteome, 166 identified as essential for pathogen survival while 26 as potential and unique therapeutic targets. Finally, 3-dehydroquinate dehydrogenase was proposed as a potent drug target for further therapeutic candidate identification. Moreover, the molecular docking and dynamic simulation technique were applied to performed a virtual screening of natural product libraries (i.e., TCM and Ayurvedic compounds) along with 3-amino-4,5-dihydroxy-cyclohex-1-enecarboxylate (DHS) as a standard compound to validate the study. Consequently, Argeloside I, Apigenin-7-O-gentiobioside (from Ayurvedic library), ZINC85571062, and ZINC85570908 (TCM library) compounds were identified as potential inhibitors of 3-dehydroquinate dehydrogenase. The study proposed new compounds as novel therapeutics, however, further experimental validation is needed as a follow-up.Communicated by Ramaswamy H. Sarma.